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Nutrients 2012, 4, 1898-1944; doi:10.

3390/nu4121898
OPEN ACCESS

nutrients
ISSN 2072-6643
www.mdpi.com/journal/nutrients
Review

Molecular Nutrition ResearchThe Modern Way Of


Performing Nutritional Science
Frode Norheim 1, Ingrid M. F. Gjelstad 1, Marit Hjorth 1,, Kathrine J. Vinknes 1,,
Torgrim M. Langleite 1, Torgeir Holen 1, Jrgen Jensen 2, Knut Tomas Dalen 1,
Anette S. Karlsen 1, Anders Kielland 1, Arild C. Rustan 3 and Christian A. Drevon 1,*
1

Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine,


University of Oslo, P.O. Box 1046, Blindern, N-0317 Oslo, Norway;
E-Mails: frode.norheim@medisin.uio.no (F.N.); i.m.f.gjelstad@medisin.uio.no (I.M.F.G.);
marit.hjorth@medisin.uio.no (M.H.); kathrine.vinknes@medisin.uio.no (K.J.V.);
t.m.langleite@medisin.uio.no (T.M.L.); torgeir.holen@medisin.uio.no (T.H.);
k.t.dalen@medisin.uio.no (K.T.D.); anette.karlsen@medisin.uio.no (A.S.K.);
anders.kielland@medisin.uio.no (A.K.)
Department of Physical Performance, Norwegian School of Sport Science, P.O. Box 4014,
Ullevl Stadion, N-0806 Oslo, Norway; E-Mail: Jorgen.Jensen@nih.no
Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo,
P.O. Box 1068, Blindern, N-0316 Oslo, Norway; E-Mail: arild.rustan@farmasi.uio.no
These authors contributed equally to this work.

* Author to whom correspondence should be addressed; E-Mail: c.a.drevon@medisin.uio.no;


Tel.: +47-22851392; Fax: +47-22851393.
Received: 6 September 2012; in revised form: 25 October 2012 / Accepted: 12 November 2012 /
Published: 3 December 2012

Abstract: In spite of amazing progress in food supply and nutritional science, and a
striking increase in life expectancy of approximately 2.5 months per year in many countries
during the previous 150 years, modern nutritional research has a great potential of still
contributing to improved health for future generations, granted that the revolutions in
molecular and systems technologies are applied to nutritional questions. Descriptive and
mechanistic studies using state of the art epidemiology, food intake registration, genomics
with single nucleotide polymorphisms (SNPs) and epigenomics, transcriptomics,
proteomics, metabolomics, advanced biostatistics, imaging, calorimetry, cell biology,
challenge tests (meals, exercise, etc.), and integration of all data by systems biology,

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will provide insight on a much higher level than today in a field we may name molecular
nutrition research. To take advantage of all the new technologies scientists should develop
international collaboration and gather data in large open access databases like the
suggested Nutritional Phenotype database (dbNP). This collaboration will promote
standardization of procedures (SOP), and provide a possibility to use collected data in
future research projects. The ultimate goals of future nutritional research are to understand
the detailed mechanisms of action for how nutrients/foods interact with the body and
thereby enhance health and treat diet-related diseases.
Keywords: molecular nutrition; nutrigenomics; genomics; transcriptomics; proteomics;
metabolomics; systems biology; adipokines; myokines

1. Introduction
Mankind has gone through several revolutions concerning dietary habits. A large break-through was
the mastering of fire to be able to cook or fry foods making nutrients more bioavailable in particular to
children. This more systematic use of fire related to butchering took place in several places of the
world in parallel about 400,000 to 200,000 years ago [1,2]. Another striking period of human history is
represented by the Neolithic Revolution with development from a foraging life-style (gathering and
hunting) mostly associated with nomadic activities, to an agricultural activity of people settled in
permanent areas of fertile land. Early activity of this type is described in particular along large rivers
(Euphrates, Tigris and Nile; [3]). Introduction of fire and agriculture made it possible to feed more
people and are prerequisites for development of the amazing expansion of the global population from
about 5 million people 10,000 years ago up to 7 billion people today [4]. These few examples of
nutrition illustrate the obvious fact that healthy and sufficient food is essential for population growth.
1.1. Population Growth and Life Expectancy
Throughout most of human history, the pace of growth of the global population has been very
slow [5]. World population reached 1 billion around 1800, and after another 125 years it was 2 billion.
The world is currently in a period of faster population growth, increasing from 3 to 7 billion within the
space of the past half-century [6]. In 2011, there was ~135 million births and 57 million deaths, a net
increase of 78 million people [4]. According to the latest medium-fertility projections the world
population will continue to grow throughout this century, reaching 9.3 billion in 2050 and 10.1 billion
in 2100, obviously with a large degree of uncertainty. With the present trends there is a marked
increase in age of most populations, both in the group of working age (1565 years) and in the elderly
(above the age of 60 years).
During the last 150 years the life expectancy has increased by about 32 years in Norway for boys
born in 2011 up to 79 years, while the life expectancy has increased by about 34 years for girls up to
83 years [7]. A similar trend is seen internationally with an increase during the last century of about
2.5 months per year. This striking increase in life expectancy in several countries is probably due to

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factors like improved quality of drinking water, more effective sewer systems, improved personal
hygiene, effective vaccination programs and improved diet.
1.2. Modern Nutrition Research
In spite of the fact that life expectancy has increased markedly during the last few centuries all
populations may benefit from optimized nutrition to reduce incidence of obesity, type 2 diabetes
mellitus (T2D), cardiovascular diseases as well as several types of cancers and infectious diseases.
Nutritional science should be focused on preventing development of diseases as well as supporting the
repair processes important for curing already fully developed diseases [8]. Traditional nutrition
research has contributed significantly to modern biomedicine and obviously promoted prolonged life
expectancy. However, it is still a large potential for improving diet and health for many groups in
economically developing as well as developed countries. This potential can be exploited by designing
good studies and applying new and advanced techniques, mostly based on molecular methods and
advanced biostatistics.
1.3. Nutrigenomics and Molecular Nutrition
The National Institute of Health defines Genomics as the study of all of a persons genes, including
interactions of those genes with each other and with the persons environment [9]. Genomics includes
the scientific study of complex diseases such as T2D and cancer because these diseases are typically
caused more by a combination of genetic and environmental factors (genetic interactions and
gene-environment interactions) than by individual genes. Nutrigenomics is by definition a broader
field of science than genomics. It is the study of the genome-wide influence of nutrition and the
subsequent time-dependent response in transcriptomics, proteomics, and metabolomics to describe the
phenotype of a biological system [10,11].
The concept of nutrigenomics has been introduced in particular in association with the
establishment of the FP6 Network of Excellence named just Nutrigenomics Organisation [12], and
with the publication of Mller & Kerstens paper Nutrigenomics: Goals and Perspectives [10]. The
concept of nutrigenomics has often been focused on the effects of nutrients and other food constituents
on gene expression, in particular as ligands for transcription factors exemplified with the discovery of
the nuclear receptors retinoid X receptors (RXRs) and peroxisome proliferator-activated receptors
(PPARs) with retinoic acid and fatty acids (FAs) as ligands, where nutrients like FAs and derivatives
of retinol can alter transcription of DNA to RNA. The influence of genetic variation on absorption,
metabolism, elimination or biological effects of nutrients have also traditionally been included in the
concept of nutrigenomics to optimize nutrition according to the subjects genotype.
Although several great discoveries have been described with the nutrigenomic approaches, the
understanding of how nutrients execute their biological effects also depends on mechanisms not acting
through the genome (Figure 1). The concept of molecular nutrition research is broader than
nutrigenomics, and may be defined as Science concerned with the effect of nutrients and foods/food
components on whole body physiology and health status at a molecular and cellular level. The precise
determination of molecular mechanisms underlying human health and disease offers a great potential
for promoting health, and lowering mortality and morbidity, and includes the science of nutrigenomics.

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Figure 1. Molecular mechanisms of nutrients exemplified by fatty acids.

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In this review we will focus on old and in particular new technologies to describe the mechanisms
of action by which nutrients execute their biological functions under different physiological conditions.
It is a great potential for improving health by understanding the interaction between nutrients/foods
and body functions, and thereby improve dietary prevention and treatment of diseases affecting people
in affluent as well as poor societies.
2. Nutrition and Metabolism Are Complicated
2.1. Nutrition Is Demanding
In order to improve nutritional knowledge we have to consider the complexity of nutrition as
outlined in Table 1.
Table 1. Nutrition is complicated due to many variables.

We eat ~1.5 kg food & drink ~2 L liquid/day;


About 40 essential nutrients are known;
Thousands of known compounds in foods without known biological functions;
Thousands of unknown compounds in foods without known biological functions;
About 1013 cells in the body & about 1014 bacteria in the GI tractus;
Mostly unknown and complicated interplay between diet and the microbiome;
Many organs & some hundreds of cell types are found in the body;
About 25,000 genes in human cells;
Human genome includes 3 billion base pairs;
Some millions single nucleotide polymorphisms (SNPs);
A large epigenetic variation between individuals due to environmental factors;
About 100,000 transcripts (mRNA);
About 100,000 proteins;
About 1000 lipids & thousands of water-soluble metabolites.

Nutrition is complicated because of the multitudes of essential nutrients, known and unknown
chemical compounds without known biological functions, different cell types and the extensive
microbiological activity in the intestine, combined with a great genetic and epigenetic variation. All
the variable factors allow an extensive variation between individuals as well as between different
physiological states like fasted, fed, cold, warm, rested, exercised, exhausted, male and female,
menstrual cycle, pregnant, lactating and age ranging from newborn to old. This extensive complexity
of nutritional science demands advanced approaches to unravel the relations between diet and health
for different ages, sexes and environmental conditions.
2.2. Fatty Acid Metabolism Is Complex
To illustrate the complexity of nutrition research we will describe some aspects of metabolism and
mode of action of FAs (Figure 1).
An adult consumes approximately 85 g of triacylglycerol (TAG) daily. During digestion, free FAs
(FFAs) and monoacylglycerols are released and absorbed in the small intestine. In the intestinal

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mucosa cells, FFAs are reesterified to TAG, incorporated in chylomicrons, transported to systemic
circulation, and hydrolyzed to FFAs in capillaries mainly in muscle and adipose tissue.
FFAs enter cells mainly by FA transporters in the plasma membrane and are bound to FA-binding
proteins (FABP) (Figure 2), activated to acyl-CoA before they are shuttled via acyl-CoA-binding
protein (ACBP) to mitochondria or peroxisomes for oxidation to form ATP and heat, or to
endoplasmic reticulum for esterification to different classes of lipids such as phospholipids, cholesteryl
esters and TAG. FFA stored as TAG in lipid droplets may undergo lipolysis and reesterification.
Glucose may be transformed to FFA (de novo lipogenesis) if there is a surplus of glucose/energy in the
cells (Figure 2).
Figure 2. Simplified view of fatty acid metabolism.

2.3. Ligands for Transcription Factors/Altered Gene Expression


FAs or their derivatives (acyl-CoA or eicosanoids) and phospholipids may interact with nuclear
receptor proteins that bind to certain regulatory regions (promoter) of DNA and thereby alter
transcription of these genes (Figure 1) [13]. The receptor protein may in combination with a FA
function as a transcription factor. The first example described of this was PPAR. FAs as well as
eicosanoids can bind directly to PPAR and PPAR [14,15]. Strong activators of PPAR and PPAR
are unsaturated FAs such as oleic acids, linoleic acid (18:2, n-6), alpha-linolenic acid (18:3, n-3) and
arachidonic acid (AA, 20:4, n-6). FAs may also influence expression of several glycolytic and
lipogenic genes independent of PPAR. Polyunsaturated FAs (PUFA) may influence proliferation of

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white blood cells along with the cells tendency to die by programmed cell death (apoptosis) or necrosis.
Thus, FAs may be important for regulation of gene transcription and thereby regulate metabolism, cell
proliferation and cell death.
2.4. Eicosanoids
Eicosanoids are signal molecules formed from 20 carbon atoms PUFA derived from the diet. The
most common precursor for eicosanoids is AA. These multitudes of signal molecules are called
leukotrienes, prostaglandins, thromboxanes, prostacyclins, lipoxins and hydroxy-FAs. Eicosanoids are
important for several cellular functions like platelet aggregability, cellular chemotaxis and cell growth.
Eicosanoids are mostly synthesized in cells where they execute their effects, and they are rapidly
formed and degraded. Different cell types produce various types of eicosanoids with different
biological functions. For example, platelets mostly make thromboxanes, whereas endothelial cells mainly
produce prostacyclins. Eicosanoids derived from very long-chain n-3 PUFA (mostly eicosapentaenoic
acid (EPA, 20:5, n-3) are usually less potent than eicosanoids derived from n-6 PUFA [16].
2.5. Substrate Specificity
FAs may execute their action by having a different ability to interact with enzymes or receptors, as
compared to other FAs. For example, EPA is a poorer substrate than all other examined FAs for
esterification to cholesterol [17] and diacylglycerol [18]. Some n-3 PUFA are preferred substrates for
certain desaturases [19]. The preferential incorporation of n-3 PUFA into some phospholipids, is
caused by n-3 PUFA being preferred substrates for the enzymes responsible for phospholipid synthesis.
2.6. Membrane Fluidity
When large amounts of n-3 PUFA are ingested, there is a high incorporation of these FA in
membrane phospholipids, which may alter physical characteristics of the membranes. The very large
amount of DHA in phosphatidylethanolamine and phosphatidylserine in certain areas of the retinal rod
outer segments is probably crucial for the function of membrane phospholipids in light transduction,
because these lipids are located close to the rhodopsin molecules. The flexibility of membranes from
blood cells in animals fed fish oil, is markedly increased, and may be important for the
microcirculation. Increased incorporation of very long-chain n-3 PUFA into plasma lipoproteins
changes the physical properties of low-density lipoproteins (LDL) promoting reduced melting point of
core cholesteryl esters [20].
2.7. Lipid Peroxidation
Lipid peroxidation products may act as biological signals. A major concern with intake of PUFA
has been the high degree of unsaturation and thereby the possibility of promoting peroxidation of LDL.
Modified LDL may be endocytosed by macrophages and initiate development of atherosclerosis.
Oxidatively modified LDL has been found in atherosclerotic lesions, and LDL rich in oleic acid seems
to be more resistant to oxidative modification than LDL enriched with n-6 PUFA in rabbits. It should
be recalled that the dietary amount of saturated FAs, trans-FAs and cholesterol are the lipids that

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strongly correlate to development of coronary heart diseases, whereas the amount of dietary PUFA is
related to reduced incidence of these diseases. Several studies suggest it is important to have the proper
amount of antioxidants with the PUFA to decrease the risk of lipid peroxidation [21].
2.8. Acylation of Proteins
Some proteins are acylated with stearic (18:0), palmitic (16:0) or myristic acid (14:0) [22], thereby
influencing anchoring or folding of certain proteins, which may be crucial for the function of these
proteins. Although the saturated FAs are most commonly covalently linked to proteins, also PUFA
may acylate proteins [23].
A few aspects of FA metabolism in cells have been focused in this section (Figure 2) but
an extensive complexity of FA metabolism can be illustrated by the network of associations outlined in
Figure 3. In this figure multivariate analysis identifies a strong relationship between dietary n-3 PUFA,
adipose tissue gene expression and markers of metabolic health [24].
Figure 3. Network of associations between dietary intake, adipose gene expression, and
phenotypic markers. Green nodes: nutrients; yellow: lipid, fatty acid, and apolipoprotein
variables in blood; red: inflammatory and oxidative stress markers in blood; blue: gene
expression (enzyme) in adipose tissue. Solid line: positive correlation/covariance; dashed
line: negative correlation/covariance. Note: This figure is adapted with permission
from [24], Copyright 2011 Morine et al.

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3. Methods in Modern Nutrition Research


Nutrients may influence gene expression directly as ligands for nuclear receptors or by inducing
epigenetic modifications. However, nutrients are also essential building blocks (essential amino acids),
may act as coenzymes in chemical reactions (vitamins), can be converted into bioactive products (fatty
acids), inhibit oxidation of other molecules (antioxidants), or serve as energy sources.
New and advanced molecular techniques provide opportunities in nutritional science. These
technologies are often based on the different omics (genomics, epigenomics, transcriptomics,
proteomics and metabolomics) (Figure 4). Some molecular methods, which can be applied in nutrition
research (Table 2) will be put in context and explained in the sections below.
Figure 4. Dietary factors may interact with multiple biological processes. (Genomics)
nutrients interact with genes and alter functional outcomes like dietary treatment of
phenylketonuria; (epigenomics) nutrients may induce epigenetic changes like fatty acids
promote methylation of the PGC-1 promoter; (transcriptomics) nutrients may influence
gene expression as ligands for nuclear hormone receptors; (proteomics) nutrients
may post-translationally modify proteins, e.g., protein-energy malnutrition leads to
post-translational modifications of transthyretin; and (metabolomics) nutrients may change
the metabolomic signature in the blood, e.g., carotenoids are biomarkers of fruit and
vegetable intake.

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Table 2. Methods in nutritional research.

Research Area
Epidemiology
Genomics
Epigenomics
Transcriptomics
Proteomics

Metabolomics

Microbiota

Imaging

Calorimetry
Cognition

Systems biology

Technologies
Observational
Experimental
Microarray
Next generation sequencing
Bisulfite sequencing
ChiP-sequencing
Microarray
RNA sequencing
Chromatography
Electrophoresis
Mass spectrometry
Protein microarrays
Gas liquid chromatography
Liquid chromatography
Mass spectrometry
Nuclear magnetic resonance
Sequencing the 16S rRNA gene
Metaomics (includes all omics
described above)
CT
MRI
PET
SPECT
Optical imaging
Indirect calorimetry
Direct calorimetry
Cognitive tests (K-ABC, Fagan,
ERP, Kendrick object learning,
Trail making, Digit symbol,
Block design, Mini-mental state
examination, Oral word
association), EEG
Mathematical modeling
Statistical methods

Assessed Parameters
Association between diet and health outcomes
and effects of controlled dietary changes
Association between genetic variation
(e.g., SNPs, alleles) and phenotypic traits
DNA methylation and histone modification
mRNA levels and splice variants
Protein composition and
posttranslational modifications

Metabolites

Microbe species composition; genome,


transcriptome, proteome and metabolome of
the microbiotic community
Whole body dynamic non-invasive detection of
body composition (fat and lean mass), gene
regulation and molecular tracers and probes

Energy intake and expenditure


IQ (sequential & simultaneous processing,
nonverbal abilities, recognition memory)

Integrate large data sets to understand complex


physiological systems

SNP: Single nucleotide polymorphism; ChIP: chromatin immunoprecipitation; CT: computed tomography;
MRI: nuclear magnetic resonance imaging; PET: positron emission tomography; SPECT: single photon
emission computed tomography; K-ABC: Kaufman assessment battery for children; ERP: evoked response
potentials; EEG: electroencephalography.

3.1. Nutritional Epidemiology


Epidemiology is the study of determinants and occurrence of disease in human populations [25].
The main objective in nutritional epidemiology is to study the role of nutrition in causes and prevention
of disease to ensure the highest quality of health recommendations [26,27]. Epidemiological research

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plays an important complementary role to experimental investigations in animals and in vitro, and can
be used effectively to generate hypothesis for mechanistic studies [25,28].
The key advantage of nutritional epidemiology is its direct relevance to human health, in contrast to
findings from in vitro studies and animal experiments, which cannot be extrapolated directly to
humans. However, the methods for collecting data on food intake are inaccurate and not easily
reproducible [25,26]. Thousands of foods with different origin and composition are consumed in the
modern societies. Although individual food habits are more limited than for the whole population,
there is a big variation from day to day and seasonal, in addition to a significant change through the
different stages of life [29,30]. No matter what methods are used for collection of dietary intake like
24 h recall, food frequency questionnaires or weighed food registration, we have serious challenges
with under- and over-reporting or adjustments of food intake due to the data collection. Moreover,
interpretation of data from epidemiological studies can be difficult because bias and confounding
factors may affect the results [25,26]. Determining causality is impossible in observational
epidemiological studies, whereas experimental studies give stronger evidence for causality. The
disease process is often complex, and multiple risk factors may interact in development of disease.
Observational epidemiology is primarily used to obtain disease information, measure prevalence and
develop hypothesis about disease etiologies. Experimental epidemiology is focused on testing
hypothesis and establishing the effect of dietary changes on health outcomes.
Identification of a link between an exposure and health outcome often begins with an
epidemiological study. An example is the association between obesity and elevated concentrations of
several plasma amino acids. The type of dietary protein turns out to be associated with the risk of
obesity, suggesting that specific amino acids may contribute to regulation of body weight [3133].
Notably, in large epidemiologic studies, plasma total concentration of the sulfur-containing amino acid
cysteine (tCys) is strongly and independently associated with fat mass and odds of obesity in adult
populations [34,35]. Recently, the cysteine-fat mass relationship has been confirmed in younger
subjects. Plasma tCys was associated with body fat percent and obesity in 984 children and adolescents
(419 years) [36], and with waist circumference in 677 prepubertal children (6 to 11 years) [37].
However, because these findings are derived from non-experimental studies (Figure 5), interpretation
must be performed carefully; high plasma concentration of tCys might promote obesity or obesity may
influence cysteine metabolism and raise plasma tCys. Another possibility is that confounding factors
may increase tCys and predispose for obesity, or that tCys might be a marker associated with obesity
or obesity-related morbidity.
To further clarify the molecular pathways and mechanisms linking cysteine and obesity, in vitro and
animal studies have been carried out. Earlier in vitro studies have demonstrated that cysteine stimulates
de novo lipogenesis and inhibits lipolysis [38,39]. Dietary cysteine supplementation decreases
metabolic rate, induces lipogenic enzymes and increases adiposity in rodents, whereas dietary
restriction of the precursor methionine has opposite effects [40,41]. This is consistent with human
studies observing that vegetarian diets (low in methionine) are associated with low weight gain [32]
and T2D risk [42]. Further evidence that cysteine is causally related to fat mass comes from studies in
rodents as well as humans showing that genetic enzyme defects increasing or decreasing cysteine
formation increase or reduce body weight, respectively [43,44]. Moreover, epidemiological and cell
biological data suggest a redox mechanism, possibly via H2O2 signaling pathways [38,45], although

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this needs further investigation. Thus, cellular, animal and epidemiological data point to an obesogenic
action of cysteine [46], although more research is required before we can conclude that cysteine is
important for development of obesity.
Collective data from cellular, animal, and human studies are required to identify mechanisms,
consequences and importance of potential links between the exposure and outcome, as illustrated by
the possible involvement of cysteine in human obesity. No epidemiological study can alone provide an
absolute answer about the effect of the exposure on the outcome [28]. When an association between a
risk factor and the outcome is supported by evidence from a large number of observational studies,
basic sciences about biological mechanisms, and experimental epidemiology, causality is strengthened
and dietary guidelines may be provided [27] (Figure 5).
Figure 5. Epidemiological methods related to other studies in nutritional science.
Observational epidemiology includes cohort, case-control and cross-sectional studies,
whereas experimental epidemiology includes field trials, community trials and intervention
studies. Observational studies help formulate hypothesis to be tested in subsequent
experimental studies. Mechanistic studies are important for understanding physiological
and biological mechanisms at cellular, tissue, and whole body level. When evidence is
supported by a large number of data from in vitro, animal, and epidemiological studies
dietary recommendations can be made.
Observational
epidemiology
Crosssectional

Non-experimental studies
Hypothesis generation

Casecontrol

Cohort

Experimental and
mechanistic studies
Hypothesis generation
and testing

Experimental
studies

Experimental
epidemiology

In vitro
studies

Field trials

Animal
studies

Community
trials

Dietary
recommendations

Intervention
studies

3.2. Genomics
The suffix -ome comes from the Greek for all, every, or complete. Genomics refer to the study of
all the genes (the genome) of an individual, including interactions of those genes with each other and
with the individuals environment [9]. Strictly speaking, genomics does not include transcriptomics
and epigenomics. Thus, in this section we focus on the interaction between diet and the genome and
present separate sections on epigenomics and transcriptomics.

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The physiological effect of a nutrient depends on multiple processes such as digestion and
absorption in the gastrointestinal tract, transport in the blood, uptake and metabolism in a variety of
cells, and excretion via the kidneys and gastrointestinal tract. Each of these processes involves multiple
gene products with polymorphisms, which potentially can alter the hosts physiological response to diet.
Single nucleotide polymorphisms (SNPs) are defined as variations in DNA sequence where one of
the four nucleotides is substituted for another [47]. SNPs may either have no consequence or a
significant effect on the function of the gene product. Human genome-wide association studies
(GWAS) take advantage of the inter-individual differences due to genetic polymorphisms by
examining the statistical association between millions of SNPs in a large population and the
phenotypic trait of interest [48]. Recent GWAS have provided many loci implicated in the
development of chronic diseases such as T2D [49]. However, these loci explain only a small fraction
of the total genetic component. For example, obesity has an estimated heritability of about 65%,
whereas large human GWAS explain less than 3% of the genetic component [50].
Identifying the relevant genes contributing to complex diseases is difficult, because several genes
with small effects contribute to overall heritability. Moreover, GWAS alone does not have sufficient
power to demonstrate interactions among genes or between genes and the environment, and it is
difficult to move from locus to disease pathway directly in humans [51]. To simplify genetic analysis,
natural variations relevant to disease have been studied in rodents. This has traditionally involved
linkage-mapping methods with crosses between strains to identify quantitative trait loci (QTLs). The
advantage of performing GWAS in inbred strains of mice is that we can control environmental
exposure, select strains with large phenotypic variations, and map genes for complex traits with high
resolution compared to linkage mapping [52]. An association-based approach called the hybrid mouse
diversity panel, using more than hundred classical inbred strains, has the potential to identify
gene-environment interactions and to map genes with a resolution of less than a megabase [53].
Recent follow-up studies to large human GWAS have been designed to show gene-diet interactions.
The studies were focused on if specific dietary compounds might modulate the phenotypic effect of a
certain genetic variant. A classical example of gene-diet interactions is the dietary treatment of
phenylketonuria (PKU). PKU is a genetic disorder caused by a mutation in the gene encoding the
hepatic enzyme phenylalanine hydroxylase. This enzyme is required to metabolize phenylalanine into
tyrosine. In PKU the enzyme is absent, and too much phenylalanine accumulates in the body causing
mental retardation. However, when newborns are diagnosed with PKU they can get a phenylalanine-free
diet, which prevents the neurotoxic effects of high blood levels of phenylalanine [54]. The FTO
(fat mass and obesity associated) gene is a good example on how variation in gene sequence interacts
with environmental factors to determine phenotype, because carriers of one or more risk alleles have
a 1.5 kg higher body weight per allele [55,56]. Although the absolute effect size is modest, it should
not be underestimated at a population level, as the association potentially may influence body weight
of up to half of the worlds population [57]. Observational studies suggest that high dietary saturated
fat intake accentuate the susceptibility for obesity in carriers of the FTO risk allele [58,59].
Furthermore, a recent meta-analysis showed a 27% attenuation of the association between FTO risk
allele and the degree of obesity in physically active adults, highlighting the importance of physical
activity at least in some individuals predisposed to obesity [60].

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Thus, it is important to know how different genotypes may interact with environmental factors to
understand the effect of genetic polymorphisms on health.
3.3. Epigenomics
One of the first assumptions of adverse environmental impact on normal fetal development was
described by Barker and Osmond [61], often referred to as Barker hypothesis. By using epidemiological
data they linked low birth weight with increased risk of death from cardiovascular disease [61].
Several other studies have made similar observations associating early adverse conditions with
metabolic dysfunction later in life; the Dutch famine cohort showed increased risk of cardiovascular
diseases 45 decades later among children born to mothers who experienced extremely severe
undernutrition during the first trimester of pregnancy [62]. These interesting epidemiological data in
addition to major advances in the field of genetics led to a new research field investigating the
connection between epigenetic modifications and environmental effects such as dietary intake.
Epigenetic refers to modifications impacting gene expression occurring without changes in nuclear
DNA base sequence [63]. Epigenomics can be defined as the study of the complete set of epigenetic
modifications in a cell or a tissue at a given time.
The human body consists of more than 200 different cell types with the same DNA sequence but
unique gene expression patterns. The difference in gene expression between the cells is mainly
governed by epigenetic modifications, including changes in DNA methylation and histone
modification. DNA methylation is one of the major epigenetic modulators [64]; it can suppress gene
expression by modulating the access of the transcript machinery to the chromatin or by recruiting
methyl-binding proteins [65]. Because DNA methylation is mitotically stable, the assumption has been
that environmental factors were unlikely to induce significant changes in DNA methylation pattern in
normal adult tissues. However, recent studies support the notion that environmental factors affect
metabolic functions through epigenetic modifications.
Twin studies have shown that DNA methylation profiles were more divergent in older twins than in
infant twin pairs, suggesting that environmental factors may influence the epigenome [66].
Diet-induced weight loss for 8 weeks in obese men altered DNA methylation in peripheral blood
mononuclear cells of specific genes [67]. Changes in DNA-methylation levels among humans with
metabolic diseases were associated with alterations in expression of genes involved in mitochondrial
function, including PGC-1 [68]. Reduced PGC-1 activity is linked with the pathogenesis of
metabolic diseases as it increases metabolic and cardiovascular risk and precedes the development of
T2D [69,70]. Interestingly, whereas palmitate and oleate can acutely induce methylation of the
PGC-1 promoter, exercise induces hypomethylation of PGC-1 in skeletal muscle [71,72]. The
hypomethylation of the PGC-1 promoter in response to exercise was paralleled with an increase in
PGC-1 mRNA content [72].
Transgenerational epigenetic inheritance refers to phenotypes present in successive generations
caused by epigenetic modifications passed via the gametes [73]. Until recently, epigenetic modifications
have been considered erased during gametogenesis or early embryogenesis. However, novel findings
have shown that epigenetic marks are not always cleared between generations [74]. A well-studied
model of transgenerational epigenetic inheritance is the Agouti mice [63]. The groundbreaking study

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of Wolff and colleagues showed that methyl-supplementation of the maternal diet induced epigenetic
regulation and altered the Agouti gene expression in the offspring causing altered fur color [75].
A striking example of transgenerational effect on metabolic disease was published by Ng et al. [76].
They showed that when male rats were fed a high-fat diet before mating their female offspring
inherited programmed -cell dysfunction. This phenotype was associated with variation in the
methylation pattern of the Il13ra2 gene. In humans, the epigenetic state at birth may predict later
childhood adiposity [77]. In two independent cohorts greater methylation of RXRA at birth was
strongly correlated with larger adiposity in later childhood. Furthermore, early carbohydrate intake
during pregnancy was inversely associated with childhood adiposity [77].
The study of microRNAs is often classified to be part of epigenetics. MicroRNAs are small
non-coding RNA molecules derived from hairpin precursors, usually between 20 and 30 nucleotides in
length [78]. They can mediate post-transcriptional silencing for about 30% of protein-encoding genes
in mammals by pairing with complementary sites in the 3 untranslated regions of target genes.
Interestingly, a recent study showed that exogenous plant food microRNAs can regulate target genes in
mammals [79]. Zhang et al. [80] showed that MIR168a, a plant microRNA, may pass through the
gastrointestinal epithelium and enter the blood and organs. Furthermore, they showed by in vitro and
in vivo studies that MIR168a can bind to the human/mouse LDL receptor adaptor protein 1 (LDLRAP1)
mRNA, inhibit hepatic LDLRAP1 expression, and reduce LDL removal from plasma.
In conclusion, several lines of evidence indicate that some of the effects of diet and physical activity
are induced via epigenetic modifications.
3.4. Transcriptomics
Transcriptomics refers to the complete collection of gene transcripts in a cell or a tissue at a given
time [10], and may be used to study gene transcription in response to dietary changes [81]. The nuclear
hormone receptor superfamily of transcription factors is probably the most important group of nutrient
sensors, which influence gene expression. Numerous nuclear hormone receptors, such as RXR,
PPARs, and liver X receptor (LXR), bind nutrients and undergo a conformational change that results in
the coordinated dissociation of co-repressors and the recruitment of co-activator proteins to enable
transcription activation.
RNA microarray technologies and sequencing can be used to evaluate the interactions between diet
and genes measured as changes in genetic expression. When applied together with traditional
biochemical methods, transcriptomics provide more extensive information about nutrition status and
metabolic responses to diet. Transcriptomics is mainly used for three different purposes in nutrition
research (reviewed in [10]); first, it can provide information about the mechanism underlying the
effects of a certain nutrient or diet; second, transcriptomics can help to identify genes, proteins or
metabolites that are altered in the pre-disease state and might act as molecular biomarkers; third,
transcriptomics can help to identify and characterize pathways regulated by nutrients.
Human dietary intervention studies have successfully used transcriptomics to show that diet induces
alterations in gene expression [79,82]. However, an important challenge in human transcriptomics
studies is the inaccessibility of human tissues. Blood, subcutaneous adipose tissue, and skeletal muscle
are among the tissues, which can be relatively easily collected. Thus, animal studies can be good

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supplements to human studies to understand how nutrients affect gene regulation in a variety of tissues.
A good example on how transcriptomics can be used is the study of Caesar and colleagues [83]. The
authors set out to study the effects of expanding mesenteric adipose tissue in a murine model. They
performed microarray analysis on mesenteric, subcutaneous, and epididymal adipose tissues after up to
12 weeks of high fat feeding. Interestingly, they discovered that high fat feeding induced similar
reduction in subcutaneous and mesenteric adipose tissue de novo lipogenesis, whereas the gene
expression in epididymal adipose tissue was unaffected. Follow-up analysis with targeted lipidomics
and biochemical analysis showed that de novo lipogenesis was down-regulated in the distal epididymal
adipose tissue and that this specialized adipose tissue might promote elongation and desaturation of
some essential PUFA for spermatogenesis.
3.5. Proteomics
Proteomics represents the large-scale study of the entire set of proteins expressed in a given cell,
tissue, or organism at a defined time-point. Most biological functions are transmitted via proteins like
enzymes, receptors and structural components. Studying proteins directly is necessary because gene
expression levels do not always correspond to protein abundance because protein levels are determined
by regulatory input from synthesis to degradation. Secondly, pre-mRNA transcripts might give rise to
several proteins because of alternative splicing. Thirdly, subcellular localization is important for
biological effects. In addition, posttranslational modifications and interactions with other proteins or
RNA affect protein action and activity. Diet can induce post-translational modifications of proteins.
One example is the study of Henze and colleagues showing that protein-energy malnutrition leads both
to changes in transthyretin concentration in the blood and post-translational modifications of the
protein [84].
Several review articles on the use of proteomics in nutrition research have been published [8587].
The focus has been on identifying new health biomarkers and bioactive peptides in foods. Among the
potential approaches for studying the proteome in large scale, chromatography combined with
mass spectrometry (MS) has become a leading method (Figure 6). Other techniques include one- and
two-dimensional gel electrophoresis and antibody-based assays such as protein microarrays [88].
Figure 6. A common workflow in a proteomic experiment. Protein samples can be derived
from tissues, plasma, cultured cells or organelle fractions. Proteins are digested (1) and the
resulting peptides are separated by chromatography (2), ionized (3) and the mass-to-charge
ratio (m/z) is measured in an initial scan (4). To identify the amino acid sequence, peptides
are selected for fragmentation and subjected to MS/MS (5). Finally, bioinformatics tools
are used to identify and/or quantify the proteins in the sample (6).

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Current MS technology makes it possible to analyze several thousand proteins in a single


sample, and is used for identification and quantification of proteins, as well as characterization of
post-translational modifications and protein interactions [89,90]. Several approaches are used, but the
method often involves digesting proteins in the sample into peptides (e.g., with trypsin) and
fractionating the peptides (often with liquid chromatography (LC)) before subjecting the sample to MS
analysis. Peptides are ionized and the mass-to-charge ratio measured. Most often two or several mass
analyzers are used in sequence separated by a fragmentation step. This is called tandem MS, or
MS/MS. The generated spectra can be used to determine the amino acid sequence, and thereby identify
the proteins by bioinformatics tools. This approach was used in a study investigating the skeletal
muscle secretome [91]. Skeletal muscle secretes peptides in response to muscle contraction that exert
either paracrine or endocrine effects. These peptides are termed myokines, and might be involved in
mediating the beneficial effects of physical activity on health. Proteins secreted by cultured human
myotubes were identified by LC-MS analysis of the conditioned cell culture medium. Two hundreds
and thirty-six proteins were detected, and 15 of the secreted proteins had enhanced mRNA expression
in biopsies from m. vastus lateralis and/or m. trapezius of healthy individuals after 11 weeks of
strength training.
MS can also be used for relative or absolute quantification of peptides, and the different methods
either apply labeling of peptides before MS analysis or use label-free approaches based on spectral
features [90]. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) are stable isotope
containing tags that covalently bind all peptides in a sample [92]. Stable isotope labeling with amino
acids in cell culture (SILAC) is a metabolic labeling technique where cells (and their proteomes) are
labeled by growing them in medium containing heavy or light isotopes of essential amino acids [93].
Heavy isotope-labeled model organisms (such as rodents) are also available, allowing in vivo
studies [94]. These labeling techniques have in common that differentially labeled samples are pooled
and peptides sequenced and quantified simultaneously in one run.
Forner et al. used SILAC to compare the mitochondrial proteomes of white and brown adipose
tissue in mice [95]. This was achieved by comparing each tissue to a SILAC labeled control fraction
from cultured cells. Several interesting differences were found [95]. Hwang et al. used a label-free
method to analyze changes in protein abundance in skeletal muscle in relation to insulin resistance, and
found a reduced abundance of mitochondrial proteins (among others) as compared to muscle tissue of
healthy subjects [96]. To determine changes in lysine acetylation of mitochondrial proteins during
energy restriction, a study on mice used a label-free approach and found dramatic, tissue-specific
alterations [97]. Acetylation of mitochondrial proteins was primarily regulated in brown adipose tissue
and liver. In liver, MS was used to identify specific proteins with altered acetylation, and 72 candidate
proteins involved in metabolic pathways were found.
Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that is emerging in
the field of metabolomics as well as proteomics as a complement to untargeted shotgun methods [98].
This method is particularly useful when predetermined sets of proteins, such as those constituting
cellular networks or sets of candidate biomarkers, need to be measured across multiple samples in a
consistent, reproducible and quantitatively precise manner.

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In recent years there has been a striking technological progress in the field of proteomics.
Application of tryptic digestion, chromatography, MS, antibodies and bioinformatics in combination
with other biochemical techniques, opens many new opportunities in future nutritional research.
3.6. Metabolomics
Metabolomics refers to the types and concentrations of all metabolites in a biological sample.
Biological metabolites are specific products of genomic, transcriptomic and proteomic processes of the
host or external organisms, as well as intrinsic and extrinsic influence on these. The characteristics and
concentrations of all small molecules, water- as well as lipid-soluble, provide a potential for measuring
flux through all important biological pathways, and thereby allow detailed understanding of how
metabolites interact with tissue components of functional importance [99]. Metabolomics can also be
used to identify biomarkers for intake of specific nutrients and health. For example it has recently been
shown in an meta-analysis that blood concentrations of carotenoids, a biomarkers for fruit and
vegetable intake, are more strongly associated with reduced breast cancer risk than are carotenoids
assessed by dietary questionnaires [100].
Ideally, metabolomics should have the ability to provide a detailed snapshot of biological processes
at any particular point in time. In nutritional research, such an approach may provide an opportunity to
identify changes in metabolic pathways induced by nutrients or other life-style factors, to explore
relationships between environmental factors, health and disease, and to discover novel
biomarkers [101,102]. However, due to the diverse chemical nature of low-molecular metabolites,
including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols and carbohydrates,
the global, untargeted analysis represent a tough challenge. Although development of analytical
platforms enables separation, detection, characterization and quantification of a large number of
metabolites from only minor amounts of biological samples [103], targeted metabolomics are most
often used [104].
Targeted analysis, where a pre-defined set of metabolites are monitored, may be used for
assessment of single nutrients or metabolites [105], determination of subsets of metabolites, including
lipids [106], inflammatory markers [107,108] or oxidative damage [109,110]. The profiling of lipids
has developed into its own field of lipidomics, and as adversely altered lipid metabolism is an
underlying factor in a number of human chronic diseases, lipidomics has become an important tool to
identify potential novel therapeutic targets [111,112].
Although metabolomics gain increased interest in nutrition research, there are still some major
limiting factors. In untargeted metabolomics, there are many unidentified metabolites. The high
number of unknown signals makes it often difficult to extract meaningful information. Thus, there is
a great need for publically available databases for the identification of metabolites [101]. Furthermore,
the use of pattern-recognition techniques is crucial for exploring novel molecules that may serve as
biomarkers. Moreover, the data sets based on metabolomics are usually huge and multi-dimensional.
The metabolomics data should be compiled along with data on transcriptomics and proteomics,
supporting more extensive use of bioinformatics including multivariate analyses [113].

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Due to the recognized and huge intra-individual variation, there is a need for standardization of
study design, use of large study cohorts and homogenous study populations based on preliminary
phenotyping of study subjects.
3.7. Microbiota
The human gastrointestinal tract is estimated to host up to 1014 microorganisms, tenfold the number
of human cells, predominately composed of bacteria but also archaea, protozoa and fungi. Together
they make up the gut microbiota, which during normal circumstances live in a commensal or
mutualistic relationship with their host. Their central functions in immune defense and nutrition have
led investigators to designate the gut microbiota as an organ by itself [114117]. Although humans can
live with a bacteria-free intestine the microbes are crucial for human health. For example, the gut
microbiota metabolizes indigestible carbohydrates to valuable short-chain FAs; synthesize certain
vitamins; degrade oxalates and is essential in recirculation of bile acids.
Traditional in vitro cultivation has limited the research on gastrointestinal bacteria because their
normal growth environment is complex and difficult to imitate. Thus, the introduction of
gene-sequencing of the hypervariable region in the bacterial gene for 16S rRNA on amplicons from
faecal samples has markedly extended the knowledge about their species diversity [118120]. Between
500 and 1000 different species seem to occupy a single human gut, whereas the total microbiome in
humans include between 10,000 and 40,000 species. However, the majority of microbes within the
digestive tract appear to include less than 100 different species [121,122].
Development of next-generation sequencing have permitted mapping of the microbial metagenome
in humans [123]. As part of the Metagenomics of the Human Intestinal Tract project, faecal specimens
from 124 European individuals were analyzed and an average of 4.5 Gb (ranging between 2 and 7.3 Gb)
of sequence was generated from each sample [124]. Genome annotation provided a set of 3.3 million
non-redundant microbial genes and detected some 536,000 prevalent unique genes in each individual.
Furthermore, almost 40% of the genes from each individual are shared with at least half of the
individuals in the cohort. Among these are genes involved in the biosynthesis of short-chain FAs, amino
acids and certain vitamins, which all are molecules suggested to be provided by bacteria to humans.
How nutritional habits interfere with the intestinal microbiota is far from understood. It was
traditionally believed that the microbe composition was relatively unchangeable, but DNA sequence
analyses have challenged this view. Studies have clearly shown that the composition of gut microbiota
adapt during changes from breast milk to solid food and when altering the composition of ingested
macromolecules [125]. A recent evaluation of long-term dietary habits and short-term interventions
concluded that small shift in microbe composition is prevalent after only one day, but that a period of a
year have significantly more influence on the gut microbiota [126]. Changes in intake of non-digestible
carbohydrates can affect faecal microbiota composition. A three-week controlled dietary intervention
in obese males showed especially that resistant starch influenced the abundance of several dominant
phylotypes [127]. Moreover, supplementation with galacto-oligosaccharides or inulin cause increased
content of bifidobacteria in the gut [128,129]. Notably, non-responders are often observed and the
outcomes appear to be more dependent on the initial composition of the individual gut microbiota than
the dietary interventions [130].

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Whereas, metagenomics provides insight into the genetic potential of microbiota, additional
transcriptomics, proteomics and metabolomics analyses in combination with interventions and
monitoring of the host, are necessary to develop biological model systems of functional significance
for the gastrointestinal microbiota.
3.8. Imaging
Imaging in biomedicine represents a broad scientific and clinical approach usually distinguished
based on spatial resolution (microscopic to macroscopic) or types of energy detected, like ionizing
radiation, photons or sound waves [131]. Traditionally, imaging has been used to characterize
morphological and anatomical properties, but during the last decade a new and important discipline
called molecular imaging has emerged. European Society for Molecular Imaging defines molecular
imaging as the characterization of the dynamics of molecular processes in living organisms. In
comparison to omics approaches that provides comprehensive snapshots of biological indicators or
biomarkers, molecular imaging advances this information, showing non-invasively the activity of
markers and changes in location with time. The modalities in molecular imaging are positron emission
tomography (PET) and single photon emission computed tomography (SPECT) that detect - and
-radiation; nuclear magnetic resonance imaging (MRI) that detect differences in relaxation time;
photo-acoustic imaging that detect ultrasonic waves; and optical imaging that mainly record
luminescent and fluorescent light.
Body composition is highly relevant in nutritional science and both computed tomography (CT) and
MRI can distinguish between different tissue types and thus be used to reconstruct major anatomical
compartments and tissues in vivo, thus providing direct quantification of either cross-sectional area or
volume [132]. For example, MRI has been carefully validated with dissection of adipose depots in rats
fed different types of fatty acids [133,134], and the data are in accordance with each other. The quality
of the CT analysis is well established, but due to the damaging radiation risk of CT the use of MRI is
expanding. The two methods have in comparative studies shown somewhat different results, but
the use of higher magnetic field and improved MRI imaging procedures have reduced this
discrepancy [135,136]. A potential confounding factor when attempting to quantify e.g., muscle size is
infiltration of fat. However, post hoc analytical techniques that separately quantify contractile and
non-contractile compartments within muscle tissue have been developed [137].
Essentially there are two approaches used in molecular imaging: (1) administration of molecular
probes, which recognize and bind to a particular biological molecule or are activated by a specific
process (e.g., enzymatic reaction); (2) reporter genes that are expressed in response to a gene
regulatory event.
The imaging technologies PET and SPECT are based on detection of radioisotopes, which can be
used to label a broad range of biological molecules. The most obvious differences between these
two methods is that PET imaging exhibit the highest sensitivity, while the longer half-life of SPECT
emitters offers a wider observational time-window. In nutritional science the most used radio-label
probe is (18-F)fluro-D-glucose (FDG), which among others is used to explore glucose intolerance. In
optical imaging, fluorescent molecules or chemiluminescent reactions are detected. This involves
substantial structural change of labeled molecules, consequently changing the biological properties.

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However, genes encoding such molecules can stably be integrated in the genome offering the
possibility of engineering reporter constructs as in transgenic reporter mice [138]. This enables timely
unlimited studies of dietary effects on gene regulation. For example, transgenic mice reporting
NF-B activity have been used to show anti-inflammatory effects of dietary plant extracts [139].
3.9. Assessments of Energy Expenditure Using Calorimetry
Modern society has developed sophisticated methods to improve production, storage and
distribution of nutrients. Thus, malnutrition occurs rarely in developed countries, but we suffer from an
excess of energy supply. Tight control of energy intake and expenditure is essential for survival and a
healthy life. Organisms have evolved to promote anabolism to store energy when energy supply
exceeds demands and switch to catabolism when supply is lower than demand. Whereas some people
have a remarkably stable weight over time, others steadily gain weight through a constant net surplus
intake of energy. An important aspect of modern nutritional science is to identify genetic, nutrient and
environmental factors promoting a stable body weight.
Identification of leptin as a genetic determinant of the obese phenotype model (ob/ob) [140]
awakened interest in the analysis of energy expenditure in mice. Our ability to alter the genome in
mice, either by deleting genes using knock out approaches, or by over-expressing genes, often results
in mice with changes in body weight or body composition. Mouse genetics has therefore a great
potential to increase our understanding of energy metabolism. Sophisticated methods to evaluate if
food intake or energy expenditure (or both) contribute to the altered phenotype, may be used to analyze
genetically modified animals.
Energy expenditure can non-invasively be determined by direct or indirect calorimetry. Direct
calorimetry assesses energy expenditure by direct measurement of heat produced by the animal [141]
(Figure 7). The animal is placed inside an insulated chamber and the produced heat is measured using
a calorimeter. Calorimeters are expensive with slow response time and they do not provide information
about the nature of the oxidized substrates. Although direct calorimetry is the only method that can
accurately quantify heat production and metabolic rate (MR) in metabolically normal as well as
abnormal states, the technique is rarely used. With indirect calorimetry, energy expenditure is
calculated based on accurate measurement of O2 consumption and CO2 production. This type of
calorimetry has been applied in clinical settings and can be done in several ways [142]. A typical
indirect calorimetric system includes a set of gas-tight chambers, which are ventilated with a steady
flow of fresh air. Once an animal is placed inside a chamber, it will consume O2 and produce CO2. The
decrease in O2 and increase in CO2 in each chamber is calculated against a reference chamber. The fast
response time for the O2 and CO2 sensors enable sequential measurement of the chambers at 13 min
intervals. In a typical experiment, 10 cages can be measured 4 times per hour. In addition to the speed,
measurement of changes in the two gasses enables assessments of respiratory exchange ratio (RER),
which is defined by the ratio of CO2 exhaled and O2 inhaled. Metabolism of carbohydrates gives a
theoretical RER of 1.00, for protein the value is 0.83, and 0.70 for fat. Identification of the primary
substrate oxidized gives additional information about the phenotype. Due to its simple measurements,
indirect calorimetry has become the gold standard for assessment of energy expenditure. However, it is
important to be aware that indirect calorimetry relies on assumptions that have never been tested to be

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accurate for genetic or pharmacologically induced changes in metabolic fuel partitioning affecting
storage or promoting obesity and T2D [141].
Figure 7. Indirect calorimetry. The typical indirect calorimetric system includes several
gas-tight chambers (here illustrated by two housing cages and a reference cage), control
units, control system and gas bottles. During the experiment, air of similar origin is
delivered simultaneously to all chambers via the sample switch unit. Gas from each
container is sequentially sent to the air-drying unit prior to CO2 and O2 measurements. The
reduction in O2 and production of CO2 in chambers housing mice is calculated against the
measured values of these gasses coming from the reference cage. Modern O2 and CO2
sensors have fast response times of minutes, which enable measurements of 1020 cages
several times per hour. The calorimetric chambers can be combined with devices for
measurements like food and liquid intake, body weight, movement, voluntary exercise
(e.g., running wheels), temperature in the cage, body temperature, and collection of feces
and urine.

Although equipment to measure energy expenditure is available, it is far from trivial to perform
experiments giving reliable data. Indeed, there is controversy on how to analyze and interpret energy
expenditure data obtained by indirect calorimetry as recently discussed in a guide to perform energy
expenditure experiments [143]. A major challenge with energy expenditure studies is to evaluate if
food intake or energy expenditure (or both) contribute to the altered phenotype. A consistent small
alteration in energy intake or energy balance over a longer period of time may have significant effects
on body weight and body composition. Because measurements typically are performed during a short
time, identification of such small changes requires impractically large sample sizes to get enough

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statistical power. In addition, the phenotypic changes themselves pose problems. Once obesity or
leanness has developed, behavioral and metabolic alterations that originally triggered changes in body
weight may obscure or confound the processes that caused the phenotypic changes. Analysis of two
different groups of mice, with different weight or body composition poses problems with
normalization because various organs differ in metabolic rate. White adipose tissue (WAT), which is
the tissue that expands with obesity, is metabolically less active than brain or liver. However,
transformation of WAT to brown adipose tissue (BAT) enhances metabolic activity to become one of
the most active tissues in the body.
Despite the listed problems, energy expenditure measurements are widely used to evaluate changes
in energy metabolism. Energy expenditure measurements are often combined with non-invasive body
composition analysis (MRI or CT) to determine lean tissue mass (total body weight subtracted by fat
mass). Three methods have been routinely used to normalize energy expenditure data. None of these
are reliable if there are substantial differences in body weight or body composition among the
individuals tested [144]. When evaluating earlier studies, it is important to understand the biases
caused by the different normalization methods: (1) Normalization against total body weight. Increasing
body weight is mainly caused by accumulation of adipose tissue with a low metabolic rate. Hence, this
type of normalization has a tendency to give a false reduction in energy expenditure with increasing
body weight; (2) Normalization against lean body weight. This way of normalization gives a false
increase in energy expenditure. Although adipose tissue has a low metabolic rate, accumulation of
WAT increases total body energy demand; (3) Normalization against metabolic body weight. The
relationship between mammalian basic metabolic rate and body weight is proportional to body weight
to the power of 0.75 [143]. Although this scaling seems correct when comparing different species, it is
not accurate for animals within the same species with an imbalance in organ weights and altered body
composition. To overcome problems with normalization, it has been suggested to analyze and plot
energy expenditure for individuals and evaluate differences in energy expenditure among groups with
ANCOVA [143].
Due to the difficulties with assessment of energy expenditure, it is vital to link a possible difference
in energy expenditure to other types of observations. Usually, changes in energy expenditure can be
explained by alterations in food intake, absorption of nutrients, physical activity, shivering, or RER.
Altered thermogenesis can be involved in circumstances with altered UCP1/BAT uncoupling.
3.10. Systems Biology
Systems biology is a fashion-word in modern biology used to describe all aspects of a biological
system in an integrated view. The fundamental principle is that the perspective on the whole organism
will provide a more accurate view than the sum of the parts, based on the idea that a complex system
has intrinsic properties that cannot be derived directly from the additive effects of its individual
parts [51]. As of today, the most advanced approach of system biology is to integrate the information
obtained from advanced technologies to describe and predict how the whole organism will react to
certain environmental or genetic alterations. Typically, systems biology includes the information
obtained from individual studies on genetics, epigenomics, transcriptomics, proteomics, metabolomics
and functional assays including imaging, assessment of energy expenditure or the use of challenging

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test (OGTT, physical activity, intervention with different diets or meals, fasting). By extracting
biological knowledge from a variety of technologies, integrative systems biology may provide
predictive models of cells, organs, complex biochemical processes as well as entire organisms. Such
integrative information may be used for the purpose of identifying new molecular targets of dietary
exposure as well as biomarkers of disease [145].
System-based nutrition studies typically include five steps (reviewed in [51]). The first step would
be to define the system to investigate. A system could be a cell population, an organ, or an entire
organism, like experimental animals or humans. The second step would be to decide which
information (components) that should be obtained from the system. In a transcriptomics study the
authors would typically include gene expression data. The third step would be to determine how the
components interact with each other. For example, co-variation between genes can be investigated.
Fourthly, the investigators should model the dynamics of the system to understand the interactions
between its components. For example, time-dependent gene-interactions changes can be studied in
response to a high fat diet. Lastly, the model should be validated using experimental perturbations. In a
molecular nutrition study you can for example use in vitro cell systems or genetically modified mice to
validate the findings.
An important asset in advanced systems biology is the possibility to compare experimental data
extracted from diverse available databases. Ng et al. published in 2006 a collection of 150 publicly
available databases [146]. Since then, the amount of new available data has expanded rapidly. One
example on such a database is the nutritional phenotype database (dbNP), initiated by NuGO [147].
Following from this, the heterogeneity of experimental data, within and between populations, represent
one of several challenges for systems biology, and future efforts should aim at the inclusion of full
descriptions of experimental conditions upon the entering of data in public databases.
Advanced simulation tools are of great importance to process and interpret the massive amount of
data obtained in systems biology. There are numerous efforts directed at developing a human
physiome, an extensive integrative model of human physiology that can be used for hypothesis testing
as well as education [148]. The review by Ng et al. also includes an extensive list of available tools
and resources, which may be of relevance to systems biology [143].
Systems biology can be a powerful tool in nutritional research to develop targeted nutritional
strategies. However, when extrapolating results from specific studies for the purpose of understanding
the whole organism, it is of great importance to consider the organism as a highly complex system
comprising multiple feedback mechanisms to a number of inputs including dietary intake. For
example, although linear responses may be observed in experimental models, the response of the
human organism to extrinsic challenges is rarely linear and the output of individual phenotypes cannot
be derived directly from the additive effects of its individual parts. Blood pressure is an example
commonly used as a disease-related endpoint in intervention studies, which is regulated by a number
of fast-acting neural mechanisms, slow-acting hormonal mechanisms, and long-term effects of body
fluid volume and compositions. Furthermore, the physiological response is a qualitative and
quantitative function of sex, age, body composition and a number of other individual features. Finally,
the output of individual phenotypes may not easily be interpreted from one extrinsic influence.

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Thus, many high-throughput technologies applied over time, on natural genetic variation, and in
response to different extrinsic challenges needs to be integrated into a quantitative mathematical model
to fully appreciate a complex biological system.
4. Lessons Learnt from Molecular Methods Applied in Different Tissues
4.1. Adipose Tissue
Obesity has reached epidemic proportions in many developed as well as developing countries
around the world. The global increase in obesity is tightly associated with the increase in
complications such as T2D and cancers.
Energy intake and energy use is finely balanced, and an imbalance of only 2.5% over a period of
10 years will lead to the accumulation of 30 kg of fat [149]. The main challenge to combat the obesity
epidemic is to modulate dietary and exercise patterns, which is very hard as food in modern societies
is becoming cheaper, work and leisure less energy demanding, and food and food commercials, are
often seductive. On the positive side, a few % reduction in energy balance would reduce obesity
markedly [150].
In obesity most of the expansion of fat reserves occurs in the abdominal, gluteal and femoral
subcutaneous depots, in addition to large intra-abdominal depots (also called visceral fat), such as
omental, mesenteric and retroperitoneal depots. Using CT scanning for adipose depot measurement, it
has been demonstrated that visceral fat accumulation is associated with glucose intolerance,
hyperlipidemia, blood pressure and coronary artery disease [151]. Employing CT to quantify
abdominal adipose depots, a recent GWAS uncovered a new locus for visceral adipose tissue at
THNSL2 in women, but not in men [152].
In a recent population-based cross-sectional study of 5193 middle-aged and elderly men and women
from the Hordaland Health Study, the anthropometric variables that was strongly correlated with
percent body fat, in addition to BMI, were waist (r = 0.79) in men, and waist (r = 0.74) and hip
(r = 0.73) in women [153]. Importantly, visceral fat, as measured by waist circumference or waist-hip
ratio, is positively associated with risk of coronary heart disease [154], whereas hip circumference is
inversely associated with coronary disease risk [155].
Adipose depots also serve other purposes than energy storage. Some adipose depots have
mechanical functions, such as the buccal fat pad in suckling infants, the retro-orbital fat stabilizing the
eye, subcutaneous padding around the cranium, and fat pads in buttocks, hands and feet [156,157].
Adipose tissues store fat-soluble vitamins such as A and D, and alpha-tocopherol [158]. In addition,
there are insulative properties of adipose tissues, directly, but also indirectly due to a lower
surface-to-volume ratio in obesity. Furthermore, certain adipose depots provide essential FAs to
lymphoid cells [159] or sperms [83]. A combined transcriptomics, lipidomics and cell-biology analysis
of subcutaneous, epididymal and mesenteric adipose tissue reveals marked functional differences [63].
In humans, adipose tissue depots start to develop in the second trimester of pregnancy and expand
rather quickly until birth. After birth, fat depots continue to expand, by increasing adipocyte size and
number of adipocytes. Obese subjects, from 2 to 4 years of age onwards, have increased number of
adipocytes compared to non-obese children. Obese children have larger adipocytes (>0.5 g lipid/cell),

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a potentially more important parameter than adipocyte numbers; this cell size corresponds to adult size
adipocytes, which lean children do not reach until 1719 years of age [160]. Recent developments
using cell-lineage tracking, knock-in and knock-out technology in mice, have established PPAR
and C/EBP, in addition to PRDM16, as master regulators of murine adipose development
in vivo [161].
WAT serve to buffer the energy supply on a day-to-day basis. WAT respond to hormonal signals
and blood levels of nutrients, take up dietary fat after meals and release FAs for use by muscles and
heart, as carbohydrate stores are depleted during fasting.
BAT serves an important thermogenic function in human infants and animals with high
surface-to-volume ratio. The presence of BAT in adult humans has been controversial until recently,
but use of new technology, in particular PET widely used in cancer imaging, has recently demonstrated
BAT in a surprisingly high fraction of adult humans. Interestingly, BAT is stimulated by cold exposure
and varies with seasonal changes [162165]. PET studies suggest that total BAT in adults varies
between 0.5 and 170 g [162,163]. The modulation of human BAT depots can have a large potential for
treatment of obesity, because even a modest amount of activated BAT might be able to combust a
considerable proportion of daily energy intake [163]. Pharmacological modulation of BAT, via 3
adrenergic receptors, received a lot of attention in the 70s and 80s, and will probably experience new
attention from pharmaceutical companies. A novel proposition has been to reprogram human
pluripotent stem cells to BAT, which after transplantation into the body may establish functional fat
pads in vivo [166,167].
The number of adipocytes in the adult human body is remarkably stable [168]. Using DNA 14C
standard curves, based on the exponential decay of 14C originating from atmospheric nuclear bomb
tests in the early 60s, the adipocyte median turnover in humans have been estimated to be 8.4% per
year, with half-life of 8.3 years [168]. The lipids in adipocytes also are rather stable with constant
body weight, with a mean lipid half-life of 1.6 years [169]. Because ~10% of adipocytes are replaced
yearly [168], and mature adipocytes are post-mitotic [170], pre-adipocytes must differentiate into
mature adipocytes continually in adult humans. However, the identity and regulation of the
pre-adipocytes are still in question [156]. The various pre-adipocytes isolated in rodents seem to
be localized in the adipose stromal compartment, and cellular candidates are endothelial as well
as perivascular cells, whereas foetal adipocytes may share a common precursor with muscle
tissue [161,171174].
WAT is also a secretory organ that releases factors, known as adipokines, capable of regulating
several physiological processes. Two of the most known adipokines are leptin and adiponectin [175],
and the plasma levels of these adipokines are often measured with enzyme-linked immunosorbent
assay (ELISA). Plasma concentrations of adiponectin are negatively correlated with BMI, whereas
leptin increases with BMI. It is a correlation between total body fat and blood levels of leptin, with
larger adipocytes secreting more leptin [176]. Leptin, along with insulin and several other gastrointestinal
peptides, regulate satiety in the arcuate nucleus and other parts of the hypothalamus [150].
An example of how the association between different FAs and diabetes can be examined, is the use
of cultured murine 3T3-L1 adipocytes to study expression of resistin, which is an adipokine proposed
to be related to insulin resistance [177]. AA and to a smaller extent EPA reduced resistin mRNA levels
to 20% of control at 60250 M. Actinomycin D as well as cycloheximide abolished the AA-induced

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reduction of resistin mRNA levels, indicating dependence on de novo transcription and translation. The
data suggest that reductions in resistin mRNA levels involve a destabilization of the resistin mRNA
molecule, which may explain the beneficial effect of ingesting PUFAs on insulin sensitivity.
4.2. Skeletal Muscle
Skeletal muscles represent the largest tissue in healthy lean people and accounts for 40% of the
body weight in young males. Muscles have the ability to convert chemical energy to mechanical work
and are designed for contraction. However, skeletal muscles are also involved in whole body metabolic
regulation, and 70%90% of insulin-stimulated glucose uptake occurs in muscles [178]. Skeletal
muscles are likewise the main storage of glycogen and store 100600 g of glycogen, whereas the liver
only stores 50100 g in the fed state depending on the amount of dietary carbohydrates [179].
Exercise modulates metabolism of nutrients in skeletal muscles as well as the whole body [180].
The metabolic rate in skeletal muscles depends on activity level, and at rest whole body oxygen uptake
is 0.20.3 L/min, which can increase to 5 L/min in some elite athletes. For example, glucose oxidation
is about ~0.2 g/min at rest, which can increase to 3 g/min during exercise in well-trained young men.
After glycogen depleting exercise, the body will be more insulin sensitive [179].
Modern molecular nutritional science studying skeletal muscles highlights mechanisms determining
insulin sensitivity and mitochondrial biogenesis. The key signaling proteins regulating insulin
sensitivity and mitochondria biogenesis are AMPK, PGC-1, PPAR, and SIRT1 [181]. All these
signaling molecules are regulated in a complex interplay between exercise and dietary intake.
Transcriptomic analysis has shown that artificial activation of AMPK or PPAR may increase
expression of genes involved in oxidative metabolism, and pharmacological activation of AMPK can
even increase running capacity in mice [182]. Epigenetic analyses in skeletal muscles have shown that
methylation of the promoter region for PGC-1 is regulated by physical activity and decreases during
prolonged bed rest in conjunction with reduced insulin sensitivity and expression of oxidative
genes [183]. Proteomic analyses confirm that expression of a large number of oxidative enzymes is
lower in muscles of obese insulin resistant people as compared to lean subjects [184].
Gene expression and protein synthesis is increased after exercise. Importantly, amino acid
supplementation increases protein synthesis after training, and healthy muscles result from both
exercise and food intake. Global analysis of mRNA expression in skeletal muscles have shown that
amino acid supplementation after exercise regulates several genes [185]. Three hours after exercise
dietary intake of amino acids caused down regulation of genes involved in muscle contraction,
extracellular matrix, structure, proteolytic processes and signaling transduction the genes regulated by,
whereas amino acids up-regulated genes for mitochondrial FA transporters and electron transport chain
48 h after exercise [185].
Fat is an important energy substrate for muscles at rest and during exercise. FAs are supplied by
degradation of triacylglycerol stored in skeletal muscles and by uptake of FFA from blood [186]. In
obesity, triglycerides accumulate in skeletal muscles and accumulation of intermediates of fat
metabolism is believed to cause insulin resistance [187]. Furthermore, infusion of FFA increases insulin
resistance within hours [188]. A high fat diet for weeks may increase activity -hydroxyacyl-CoA
dehydrogenase (HAD) [189], but the mechanism for increasing expression of enzymes involved in

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-oxidation remains unknown in humans. However, muscles function seems to be quite resistant to a
high fat diet as long as body weight and activity level is maintained [189].
Skeletal muscles act as a major endocrine organ [91,190]. The earliest reports about secretory
proteins from skeletal muscle relate to myostatin [191] and interleukin IL-6 [192]. Myostatin was the
first recognized myokine, but major interest has been focused on IL-6, which was discovered as a
secretory product of skeletal muscle, increasing in plasma in proportion to the length and intensity of
physical activity [190]. Chronically elevated level of IL-6 is pro-inflammatory, but IL-6 produced in
muscle during exercise may act without activating classical inflammatory pathways. More recently,
Haugen et al. (2010) [193] described IL-7 as a novel myokine, which may play a role in the regulation
of muscle cell development, and IL-15 may be involved in the crosstalk between muscle and fat [194].
A 6 months exercise intervention caused hypomethylation and increased transcription of the IL-7 gene
in skeletal muscle and increased level of IL-7 in plasma [195]. Irisin is a novel myokine, which has
attracted much attention because expression is regulated via PGC-1 and irisin increases expression of
UCP1 in white adipocytes and increases thermogenesis [196]. Furthermore, exercise increases irisin in
plasma and present a potential myokine mediating beneficial effect of exercise in adipose tissue [196].
Knowledge about new myokines and their physiological functions may unravel a network of
communication between muscle and other organs.
In combination with abundant food intake, reduced physical activity is the origin for increased risk
for overweight and T2D. Unraveling the molecular mechanisms for nutritional regulation of gene
expression in skeletal muscles is a major challenge for modern molecular research and may hopefully
improve treatment of insulin resistance, although physical activity will probably remain important for
health in most organs, the brain included.
4.3. Liver
The liver is a major metabolic organ. In the fed state, insulin inhibits gluconeogenesis and promotes
glycogen synthesis and de novo lipogenesis in a healthy liver. In the fasted state, the drop in insulin
causes reduced lipid production and increased hepatic gluconeogenesis and glycogenolysis.
FAs can accumulate in hepatocytes as TAGs or they may be exported as part of very low-density
lipoproteins (VLDL). FAs exported as TAG may be reesterified and stored in adipose tissue or used as
fuel in skeletal muscles and other cells. The hepatic TAG content is regulated by the activity of cellular
proteins facilitating uptake, synthesis, esterification, and oxidation of FAs, and TAG export. Moreover,
FAs and FA derivatives regulate hepatic lipid metabolism by binding nuclear receptors that modulate
gene transcription (e.g., PPARs, LXRs, HNF-4 and SREBP) [197].
The liver also synthesizes significant amounts of cholesterol and phospholipids. Some of these are
packaged with lipoproteins and made available to the rest of the body. The remainder is excreted in
bile as phospholipids or free cholesterol, or after conversion of cholesterol to bile acids. Cholesterol
can also accumulate in lipid droplets as cholesteryl ester (CE).
To understand the mechanisms regulating hepatic lipid and lipoprotein metabolism are important in
molecular nutritional science. Non-alcoholic fatty liver disease (NAFLD) is characterized by
accumulation of TAG in hepatocytes. NAFLD may represent the hepatic manifestation of the
metabolic syndrome with visceral obesity, dyslipidaemia, and insulin resistance [198]. Eventually,

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accumulation of lipid droplets in the hepatocytes results in hepatic steatosis, which may be due to
multiple factors including increased adipose tissue lipolysis and/or high dietary energy intake
promoting an increased circulating pool of FFA and TAG. In addition, a decrease in hepatic FA
oxidation, an increased hepatic de novo lipid synthesis due to excessive conversion of proteins and
carbohydrates to TAG, and a reduced hepatic VLDL secretion may also be major determinants for
NAFLD [198,199]. Based on MRI we can obtain data on hepatic lipid accumulation, which was
unavailable previously due to ethical aspects of taking biopsies [200]. Many studies indicate
that lifestyle modifications such as weight loss and increased physical activity may reduce
hepatic steatosis [201]. Moreover, the dietary FA pattern may also be important. Supplementation with
n-3 LCPUFA appears to reduce nutritional hepatic steatosis associated with obesity in adults [202],
and compared to saturated FAs intake from butter, vegetable n-6 PUFAs has been shown to reduce
liver fat content [203].
Besides TAG and CE, other lipids like diacylglycerols (DAGs), ceramides, FAs and acyl-CoA often
accumulate in NAFLD. These lipids may interfere with hepatic function and particularly with the
ability of hepatocytes to respond to changes in insulin levels. The failure of hepatocytes to respond to
insulin by inhibition of glycogenolysis and gluconeogenesis contributes to the development of glucose
intolerance and T2D. However, most data indicate that hepatic accumulation of TAG (and CE) does
not cause hepatic insulin resistance by formation of lipotoxic FA intermediates [204]. More
comprehensive lipid analyses, including measurements of individual lipid species, and defining types
of cells and subcellular compartments in which changes in levels of specific lipids occur, may identify
new candidate lipids that are causally linked to insulin resistance. Additionally, the application of
unbiased, systems-type screens (e.g., genetics, proteomics, lipidomics, metabolomics) to the problem
may yield new theories of causation.
Like adipose tissue and skeletal muscle, the liver may also regulate peripheral insulin sensitivity
and glucose homeostasis by production of secretory proteins termed hepatokines [199]. Thus, the liver
may contribute to energy homeostasis by way of production of hepatokines and that the dysregulation
of hepatokines contributes to the pathophysiology of diabetes and subsequent complications
(e.g., NAFLD). One such liver-derived protein is selenoprotein P that induces insulin resistance and
hyperglycemia and may be involved in development of T2D [199,205].
5. Molecular Nutrition Research Applied on the Whole Organism
5.1. Different Diets
It is relatively easy to perform studies where nutrients are given as supplements, and such studies
can provide knowledge about the effect of certain nutrients. However, some studies have shown that
the biological effect of a supplement is different from the effect of foods rich in the supplied nutrient.
For example, pharmacological doses of antioxidant supplements may have harmful effects in smokers,
whereas the same amount of dietary antioxidants can be well tolerated [105]. This difference may be
because the foods rich in antioxidants also contain many other compounds affecting health. Another
explanation can be that subjects who increase their intake of a certain food product simultaneously
reduce their intake of other foods, and elimination of the other foods may cause the actual effect.

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Because nutrients and other food components act together, it is also important to do studies involving
the whole diet, like a Western diet, a Mediterranean diet, the DASH diet, a fat modified diet, etc.
We obviously do not know what nutrients or foods that promotes the biological effects, but it is very
important to define the diet to allow other scientists to reevaluate the intervention.
A challenge concerning dietary intervention is that subjects alter weight, which by itself may
influence several biological processes. Losing body weight may be a natural effect of a low fat diet.
Thus, if one is interested in the effect of modifying the fat quantity per se, and not also the effect of
changes in body weight, the diet has to be energy-adjusted with other energy containing nutrients. This
was done in the LIPGENE study where some participants got a high fat diet and others got a low
fat/high complex carbohydrate diet [206].
Although we often evaluate the effect of several dietary components, it is crucial to know what the
diet is. The best way of controlling what the participants eat is to provide all the food they shall eat
throughout the intervention [207]. However, such studies are expensive and laborious. In most studies
the participants get only part of the food they shall eat, e.g., food rich in antioxidants [105] or they get
dietary advice, possibly in combination with supply of some foods [206]. In such studies we have less
control on what has been eaten, partly because we only provide the participants with a limited amount
of food and partly because the participants may not follow the dietary advice. There are several ways
to monitor what the participants have been eating: participants can be asked about food intake during
the intervention period through questionnaires or interviews; the participants can register what they
eat, and we can collect biological samples to analyze nutritional biomarkers, i.e., objective measures of
what has actually been eaten. A good example of a valuable biomarker for marine fat intake has been
demonstrated by the strong correlation between dietary intake of marine n-3 FA and plasma n-3 marine
FA [208]. Ideally, one should have such objective biological measures for all dietary components.
However, the metabolites we can detect in biological samples often do not reflect the dietary intake of
that food component, because the component can be modified through food processing, storage,
digestion, metabolism, and the concentration of the metabolite in the biological material may not be
optimal for the food component of interest.
5.2. Challenges (Glucose Tolerance Test, Physical Exercise, Meals, Fasting)
Standardization is important to be able to compare data from different studies. The most convenient
and common way of obtaining standardized conditions is to collect samples when the research subjects
are at rest after an overnight fast. Thus, the majority of studies investigate the relatively unstressed
organism, a state perhaps not optimal for detecting markers of disease, risk or even signs of health.
Exposing the organism to a defined challenge, mimics daily life to a greater extent and may provoke
responses not seen in a resting and fasted situation. The most common challenges are physical exercise
or food intake. The hyperinsulinemic euglycemic glucose clamp is the gold standard for determination
of insulin sensitivity, but is rather labor- and time-intensive [209]. Thus, several surrogate indices have
been employed to simplify and improve the determination of insulin resistance. A prime example is the
oral glucose tolerance test (OGTT).
The OGTT is a simple method for diagnosing T2D and degrees of insulin resistance. It measures
the blood glucose concentration in response to a given oral carbohydrate load. According to the World

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Health Organization (WHO) a fasting glucose value above 7 mmol/L or a 120 min value above
11.1 mmol/L defines T2D. A 120 min value between 7.8 and 11.1 mmol/L defines impaired glucose
tolerance (IGT) [210]. For research purposes, insulin resistance indices have been used taking different
parameters into account during a glucose challenge [211,212]. Most indices based on values from
glucose challenges are more reliable than those based on fasting, as their correlation with reference
techniques is stronger [213]. There are few articles that combine molecular nutrition with glucose
challenges, such as OGTT. Shaham et al. reported changes in previously undescribed metabolites in
connection with an OGTT in healthy and prediabetic volunteers [214]. A similar approach revealed a
stronger effect of a 9 weeks anti-inflammatory drug intervention when analyses were performed on
samples taken during an OGTT-challenge as compared with the fasting state [215].
Rubio et al. describe several new catabolic metabolites as a result of extended fasting in human
volunteers [216].
A multi-challenge 4 days study including 36-h fasting, oral glucose tests, lipid tests, liquid test
meals, exercise, and cold stress, was recently reported by Krug et al. [217]. The inter-individual
variation among phenotypically similar volunteers was increased by different challenges, revealing
metabolic variation not observable in baseline metabolic profiles.
5.3. Time CoursesWhat Is the Effect of Time as Registered by Molecular Nutrition?
Intervention studies, either on nutrition or physical activity is often of limited duration, especially
when compared with the whole life-span humans sometimes use to develop a disease. Thus, a possible
effect of an intervention cannot be based only on short-term studies. For example, most people can
lose weight in the short term by reducing their intake of energy or increasing their energy expenditure.
However, few people successfully maintain their reduced body weight. One explanation for the poor
efficacy in maintaining weight loss is an active feedback mechanism linking adipose tissue to dietary
intake and energy expenditure via a set point, presumably encoded in the brain [218]. Another
explanation relates to psychological factors such as motivation to adhere to restricted regimens
diminishes with time.
Dietary intervention studies lasting up to 1 year may have a beneficial effect of a low-carbohydrate
diet on weight reduction [219]. However, a two-by-two factorial design study lasting 2 years showed
no effect of macronutrient composition on weight loss among participants advised to consume
carbohydrates with low glycemic index [220]. Two recent Swedish studies showed that a low
carbohydrate-high protein diet and a high protein diet are associated with increased incidence of
cardiovascular disease and diabetes, respectively [221,222].
Lifestyle interventions, including adoptions of a diet inducing weight reduction and increasing level
of physical activity, can prevent and reverse the development of T2D. It has recently been shown that
the benefit of lifestyle intervention extends beyond the active intervention period [223]. It was shown
in a Chinese study that a group-based lifestyle intervention lasting 6 years prevented or delayed T2D
for up to 14 years [224]. Furthermore, a Finnish study showed that most of the lifestyle changes which
reduced diabetes incidence in a population with high risk for T2D were maintained 3 years after
discontinuation of the individual lifestyle counseling [225].

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Maternal dietary intake during pregnancy and lactation is favorable for later mental development of
children. For example, some quite important studies in severely premature infants provided extra
supply of essential fatty acids in mothers milk demonstrate that cognitive function can be positively
influenced for long periods after the intervention has been performed [226].
In our present context it is essential that dietary interventions, time courses and challenges should
be combined with all the relevant molecular nutrition technologies to obtain mechanistic information
about the actual questions.
6. Conclusion
Nutritional science in the future will be heavily influenced by the new advanced methods developed
for mass measurements of genes, transcripts, proteins and metabolites, combined with advanced
imaging, epidemiology, clinical interventions with different challenges and finally bioinformatics to
integrate all information in whole body functions named systems biology. We will by this type of
scientific advancement be able to describe and sustain health, and to treat several life-style diseases
much more efficiently than we are able to do today.
Conflict of Interest
The authors declare no conflict of interest.
References
1.

2.
3.
4.
5.
6.
7.
8.

9.
10.

Karkanas, P.; Shahack-Gross, R.; Ayalon, A.; Bar-Matthews, M.; Barkai, R.; Frumkin, A.;
Gopher, A.; Stiner, M.C. Evidence for habitual use of fire at the end of the Lower Paleolithic:
Site-formation processes at Qesem Cave, Israel. J. Hum. Evol. 2007, 53, 197212.
Weiner, S.; Xu, Q.; Goldberg, P.; Liu, J.; Bar-Yosef, O. Evidence for the use of fire at
Zhoukoudian, China. Science 1998, 281, 251253.
Gupta, A.K. Origin of agriculture and domestication of plants and animals linked to early
Holocene climate amelioration. Curr. Sci. 2004, 87, 5459.
Bloom, D.E. 7 billion and counting. Science 2011, 333, 562569.
Bocquet-Appel, J.P. When the worlds population took off: The springboard of the Neolithic
demographic transition. Science 2011, 333, 560561.
World Population Prospects, the 2010 Revision. Available online: http://esa.un.org/unpd/
wpp/index.htm (accessed on 15 November 2012).
Statistics Norway. Available online: http://www.ssb.no (accessed on 15 November 2012).
Van Ommen, B.; Bouwman, J.; Dragsted, L.O.; Drevon, C.A.; Elliott, R.; de Groot, P.; Kaput, J.;
Mathers, J.C.; Muller, M.; Pepping, F.; et al. Challenges of molecular nutrition research 6: The
nutritional phenotype database to store, share and evaluate nutritional systems biology studies.
Genes Nutr. 2010, 5, 189203.
Frequently Asked Questions about Genetic and Genomic Science. Available online:
http://www.genome.gov/19016904 (accessed on 15 November 2012).
Muller, M.; Kersten, S. Nutrigenomics: Goals and strategies. Nat. Rev. Genet. 2003, 4, 315322.

Nutrients 2012, 4
11.
12.
13.
14.
15.

16.

17.

18.

19.
20.

21.

22.
23.
24.

25.
26.

1930

Afman, L.; Muller, M. Nutrigenomics: From molecular nutrition to prevention of disease. J. Am.
Diet. Assoc. 2006, 106, 569576.
Nutrigenomics Organisation Homepage. Available online: http://www.nugo.org/everyone
(accessed on 15 November 2012).
Rustan, A.C.; Drevon, C.A. Fatty Acids: Structures and Properties; John Wiley & Sons, Ltd.:
Hoboken, NJ, USA, 2001.
Hihi, A.K.; Michalik, L.; Wahli, W. PPARs: Transcriptional effectors of fatty acids and their
derivatives. Cell. Mol. Life Sci. 2002, 59, 790798.
Schoonjans, K.; Staels, B.; Auwerx, J. Role of the peroxisome proliferator-activated receptor
(PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J. Lipid Res.
1996, 37, 907925.
De Roos, B.; Mavrommatis, Y.; Brouwer, I.A. Long-chain n-3 polyunsaturated fatty acids: New
insights into mechanisms relating to inflammation and coronary heart disease. Br. J. Pharmacol.
2009, 158, 413428.
Rustan, A.C.; Nossen, J.O.; Osmundsen, H.; Drevon, C.A. Eicosapentaenoic acid inhibits
cholesterol esterification in cultured parenchymal cells and isolated microsomes from rat liver.
J. Biol. Chem. 1988, 263, 81268132.
Rustan, A.C.; Nossen, J.O.; Christiansen, E.N.; Drevon, C.A. Eicosapentaenoic acid reduces
hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme
A:1,2-diacylglycerol acyltransferase. J. Lipid Res. 1988, 29, 14171426.
Leikin, A.I.; Brenner, R.R. Microsomal delta 5 desaturation of eicosa-8,11,14-trienoic acid is
activated by a cytosolic fraction. Lipids 1989, 24, 101104.
Nenseter, M.S.; Rustan, A.C.; Lund-Katz, S.; Soyland, E.; Maelandsmo, G.; Phillips, M.C.;
Drevon, C.A. Effect of dietary supplementation with n-3 polyunsaturated fatty acids on physical
properties and metabolism of low density lipoprotein in humans. Arterioscler. Thromb. 1992, 12,
369379.
Brude, I.R.; Drevon, C.A.; Hjermann, I.; Seljeflot, I.; Lund-Katz, S.; Saarem, K.; Sandstad, B.;
Solvoll, K.; Halvorsen, B.; Arnesen, H.; Nenseter, M.S. Peroxidation of LDL from
combined-hyperlipidemic male smokers supplied with omega-3 fatty acids and antioxidants.
Arterioscler. Thromb. Vasc. Biol. 1997, 17, 25762588.
Triola, G.; Waldmann, H.; Hedberg, C. Chemical biology of lipidated proteins. ACS Chem. Biol.
2012, 7, 8799.
Muszbek, L.; Laposata, M. Covalent modification of proteins by arachidonate and
eicosapentaenoate in platelets. J. Biol. Chem. 1993, 268, 1824318248.
Morine, M.J.; Tierney, A.C.; van Ommen, B.; Daniel, H.; Toomey, S.; Gjelstad, I.M.; Gormley,
I.C.; Perez-Martinez, P.; Drevon, C.A.; Lopez-Miranda, J.; Roche, H.M. Transcriptomic
coordination in the human metabolic network reveals links between n-3 fat intake, adipose tissue
gene expression and metabolic health. PLoS Comput. Biol. 2011, 7, e1002223.
Willett, W. Nutritional Epidemiology; Oxford University Press: New York, NY, USA, 1998.
Sempos, C.T.; Liu, K.; Ernst, N.D. Food and nutrient exposures: What to consider when
evaluating epidemiologic evidence. Am. J. Clin. Nutr. 1999, 69, 1330S1338S.

Nutrients 2012, 4
27.
28.
29.

30.
31.

32.

33.

34.

35.

36.

37.

38.
39.
40.

1931

Margetts, B.; Nelson, M. Design Concepts in Nutritional Epidemiology; Oxford University


Press: New York, NY, USA, 1997.
Jeffery, E.H.; Keck, A.S. Translating knowledge generated by epidemiological and in vitro
studies into dietary cancer prevention. Mol. Nutr. Food Res. 2008, 52, S7S17.
Fowke, J.H.; Schlundt, D.; Gong, Y.; Jin, F.; Shu, X.O.; Wen, W.; Liu, D.K.; Gao, Y.T.;
Zheng, W. Impact of season of food frequency questionnaire administration on dietary reporting.
Ann. Epidemiol. 2004, 14, 778785.
Palaniappan, U.; Cue, R.I.; Payette, H.; Gray-Donald, K. Implications of day-to-day variability
on measurements of usual food and nutrient intakes. J. Nutr. 2003, 133, 232235.
Murtaugh, M.A.; Herrick, J.S.; Sweeney, C.; Baumgartner, K.B.; Guiliano, A.R.; Byers, T.;
Slattery, M.L. Diet composition and risk of overweight and obesity in women living in the
southwestern United States. J. Am. Diet. Assoc. 2007, 107, 13111321.
Rosell, M.; Appleby, P.; Spencer, E.; Key, T. Weight gain over 5 years in 21,966 meat-eating,
fish-eating, vegetarian, and vegan men and women in EPIC-Oxford. Int. J. Obes. (Lond.) 2006,
30, 13891396.
Spencer, E.A.; Appleby, P.N.; Davey, G.K.; Key, T.J. Diet and body mass index in 38,000
EPIC-Oxford meat-eaters, fish-eaters, vegetarians and vegans. Int. J. Obes. Relat. Metab. Disord.
2003, 27, 728734.
El-Khairy, L.; Ueland, P.M.; Nygard, O.; Refsum, H.; Vollset, S.E. Lifestyle and cardiovascular
disease risk factors as determinants of total cysteine in plasma: The Hordaland Homocysteine
Study. Am. J. Clin. Nutr. 1999, 70, 10161024.
Elshorbagy, A.K.; Nurk, E.; Gjesdal, C.G.; Tell, G.S.; Ueland, P.M.; Nygard, O.; Tverdal, A.;
Vollset, S.E.; Refsum, H. Homocysteine, cysteine, and body composition in the Hordaland
Homocysteine Study: Does cysteine link amino acid and lipid metabolism? Am. J. Clin. Nutr.
2008, 88, 738746.
Elshorbagy, A.K.; Valdivia-Garcia, M.; Refsum, H.; Butte, N. The association of cysteine with
obesity, inflammatory cytokines and insulin resistance in Hispanic children and adolescents. PloS
One 2012, 7, e44166.
Pereira da Silva, N.; Suano de Souza, F.I.; Ifanger Pendezza, A.; Luiz Affonso Fonseca, F.;
Hix, S.; Oliveira, A.C.; Oselka Saccardo Sarni, R.; DAlmeida, V. Homocysteine and cysteine
levels in prepubertal children: Association with waist circumference and lipid profile. Nutrition
2012, doi:10.1016/j.nut.2012.05.015.
Olefsky, J.M. Comparison of the effects of insulin and insulin-like agents on different aspects of
adipocyte metabolism. Horm. Metab. Res. 1979, 11, 209213.
Czech, M.P.; Fain, J.N. Antagonism of insulin action on glucose metabolism in white fat cells by
dexamethasone. Endocrinology 1972, 91, 518522.
Elshorbagy, A.K.; Church, C.; Valdivia-Garcia, M.; Smith, A.D.; Refsum, H.; Cox, R. Dietary
cystine level affects metabolic rate and glycaemic control in adult mice. J. Nutr. Biochem. 2012,
23, 332340.

Nutrients 2012, 4
41.

42.

43.
44.

45.

46.

47.
48.
49.

50.

51.
52.

53.

54.

1932

Elshorbagy, A.K.; Valdivia-Garcia, M.; Mattocks, D.A.; Plummer, J.D.; Smith, A.D.;
Drevon, C.A.; Refsum, H.; Perrone, C.E. Cysteine supplementation reverses methionine
restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase. J. Lipid Res.
2011, 52, 104112.
Vang, A.; Singh, P.N.; Lee, J.W.; Haddad, E.H.; Brinegar, C.H. Meats, processed meats, obesity,
weight gain and occurrence of diabetes among adults: Findings from Adventist Health Studies.
Ann. Nutr. Metab. 2008, 52, 96104.
Mudd, S.H.; Levy, H.L.; Skovby, F. Disorders of Transsulfuration; McGraw-Hill, Inc.:
New York, NY, USA, 1995.
Pogribna, M.; Melnyk, S.; Pogribny, I.; Chango, A.; Yi, P.; James, S.J. Homocysteine
metabolism in children with Down syndrome: In vitro modulation. Am. J. Hum. Genet. 2001, 69,
8895.
Furukawa, S.; Fujita, T.; Shimabukuro, M.; Iwaki, M.; Yamada, Y.; Nakajima, Y.; Nakayama, O.;
Makishima, M.; Matsuda, M.; Shimomura, I. Increased oxidative stress in obesity and its impact
on metabolic syndrome. J. Clin. Invest. 2004, 114, 17521761.
Elshorbagy, A.K.; Kozich, V.; Smith, A.D.; Refsum, H. Cysteine and obesity: Consistency of the
evidence across epidemiologic, animal and cellular studies. Curr. Opin. Clin. Nutr. Metab. Care
2012, 15, 4957.
Carlson, C.S.; Eberle, M.A.; Kruglyak, L.; Nickerson, D.A. Mapping complex disease loci in
whole-genome association studies. Nature 2004, 429, 446452.
Hirschhorn, J.N.; Daly, M.J. Genome-wide association studies for common diseases and
complex traits. Nat. Rev. Genet. 2005, 6, 95108.
Manning, A.K.; Hivert, M.F.; Scott, R.A.; Grimsby, J.L.; Bouatia-Naji, N.; Chen, H.; Rybin, D.;
Liu, C.T.; Bielak, L.F.; Prokopenko, I.; et al. A genome-wide approach accounting for body
mass index identifies genetic variants influencing fasting glycemic traits and insulin resistance.
Nat. Genet. 2012, 44, 659669.
Speliotes, E.K.; Willer, C.J.; Berndt, S.I.; Monda, K.L.; Thorleifsson, G.; Jackson, A.U.;
Allen, H.L.; Lindgren, C.M.; Luan, J.; Magi, R.; et al. Association analyses of 249,796 individuals
reveal 18 new loci associated with body mass index. Nat. Genet. 2010, 42, 937948.
MacLellan, W.R.; Wang, Y.; Lusis, A.J. Systems-based approaches to cardiovascular disease.
Nat. Rev. Cardiol. 2012, 9, 172184.
Bennett, B.J.; Farber, C.R.; Orozco, L.; Kang, H.M.; Ghazalpour, A.; Siemers, N.; Neubauer, M.;
Neuhaus, I.; Yordanova, R.; Guan, B.; et al. A high-resolution association mapping panel for the
dissection of complex traits in mice. Genome Res. 2010, 20, 281290.
Ghazalpour, A.; Rau, C.D.; Farber, C.R.; Bennett, B.J.; Orozco, L.D.; van Nas, A.; Pan, C.;
Allayee, H.; Beaven, S.W.; Civelek, M.; et al. Hybrid mouse diversity panel: A panel of inbred
mouse strains suitable for analysis of complex genetic traits. Mamm. Genome 2012, 23, 680692.
Giovannini, M.; Verduci, E.; Salvatici, E.; Paci, S.; Riva, E. Phenylketonuria: Nutritional
advances and challenges. Nutr. Metab. 2012, 9, doi:10.1186/1743-7075-9-7.

Nutrients 2012, 4
55.

56.

57.
58.

59.

60.

61.
62.

63.
64.
65.
66.

67.

68.

1933

Frayling, T.M.; Timpson, N.J.; Weedon, M.N.; Zeggini, E.; Freathy, R.M.; Lindgren, C.M.;
Perry, J.R.; Elliott, K.S.; Lango, H.; Rayner, N.W.; et al. A common variant in the FTO gene is
associated with body mass index and predisposes to childhood and adult obesity. Science 2007,
316, 889894.
Scuteri, A.; Sanna, S.; Chen, W.M.; Uda, M.; Albai, G.; Strait, J.; Najjar, S.; Nagaraja, R.;
Orru, M.; Usala, G.; et al. Genome-wide association scan shows genetic variants in the FTO gene
are associated with obesity-related traits. PLoS Genet. 2007, 3, e115.
Tung, Y.C.; Yeo, G.S. From GWAS to biology: Lessons from FTO. Ann. N. Y. Acad. Sci. 2011,
1220, 162171.
Moleres, A.; Ochoa, M.C.; Rendo-Urteaga, T.; Martinez-Gonzalez, M.A.; Azcona San Julian, M.C.;
Martinez, J.A.; Marti, A. Dietary fatty acid distribution modifies obesity risk linked to the
rs9939609 polymorphism of the fat mass and obesity-associated gene in a Spanish case-control
study of children. Br. J. Nutr. 2012, 107, 533538.
Phillips, C.M.; Kesse-Guyot, E.; McManus, R.; Hercberg, S.; Lairon, D.; Planells, R.;
Roche, H.M. High dietary saturated fat intake accentuates obesity risk associated with the fat
mass and obesity-associated gene in adults. J. Nutr. 2012, 142, 824831.
Kilpelainen, T.O.; Qi, L.; Brage, S.; Sharp, S.J.; Sonestedt, E.; Demerath, E.; Ahmad, T.;
Mora, S.; Kaakinen, M.; Sandholt, C.H.; et al. Physical activity attenuates the influence of FTO
variants on obesity risk: A meta-analysis of 218,166 adults and 19,268 children. PLoS Med.
2011, 8, e1001116.
Barker, D.J.; Osmond, C. Infant mortality, childhood nutrition, and ischaemic heart disease in
England and Wales. Lancet 1986, 1, 10771081.
Painter, R.C.; de Rooij, S.R.; Bossuyt, P.M.; Simmers, T.A.; Osmond, C.; Barker, D.J.;
Bleker, O.P.; Roseboom, T.J. Early onset of coronary artery disease after prenatal exposure to the
Dutch famine. Am. J. Clin. Nutr. 2006, 84, 322327.
Ruemmele, F.M.; Garnier-Lengline, H. Why are genetics important for nutrition? Lessons from
epigenetic research. Ann. Nutr. Metab. 2012, 60, 3843.
Handel, A.E.; Ebers, G.C.; Ramagopalan, S.V. Epigenetics: Molecular mechanisms and
implications for disease. Trends Mol. Med. 2010, 16, 716.
Cedar, H.; Bergman, Y. Linking DNA methylation and histone modification: Patterns and
paradigms. Nat. Rev. Genet. 2009, 10, 295304.
Fraga, M.F.; Ballestar, E.; Paz, M.F.; Ropero, S.; Setien, F.; Ballestar, M.L.; Heine-Suner, D.;
Cigudosa, J.C.; Urioste, M.; Benitez, J.; et al. Epigenetic differences arise during the lifetime of
monozygotic twins. Proc. Natl. Acad. Sci. USA 2005, 102, 1060410609.
Milagro, F.I.; Campion, J.; Cordero, P.; Goyenechea, E.; Gomez-Uriz, A.M.; Abete, I.;
Zulet, M.A.; Martinez, J.A. A dual epigenomic approach for the search of obesity biomarkers:
DNA methylation in relation to diet-induced weight loss. FASEB J. 2011, 25, 13781389.
Ling, C.; Poulsen, P.; Simonsson, S.; Ronn, T.; Holmkvist, J.; Almgren, P.; Hagert, P.;
Nilsson, E.; Mabey, A.G.; Nilsson, P.; et al. Genetic and epigenetic factors are associated with
expression of respiratory chain component NDUFB6 in human skeletal muscle. J. Clin. Invest.
2007, 117, 34273435.

Nutrients 2012, 4
69.

70.

71.

72.

73.

74.
75.
76.
77.

78.
79.

80.

81.
82.

1934

Mootha, V.K.; Lindgren, C.M.; Eriksson, K.F.; Subramanian, A.; Sihag, S.; Lehar, J.;
Puigserver, P.; Carlsson, E.; Ridderstrale, M.; Laurila, E.; et al. PGC-1alpha-responsive genes
involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat.
Genet. 2003, 34, 267273.
Patti, M.E.; Butte, A.J.; Crunkhorn, S.; Cusi, K.; Berria, R.; Kashyap, S.; Miyazaki, Y.;
Kohane, I.; Costello, M.; Saccone, R.; et al. Coordinated reduction of genes of oxidative
metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1.
Proc. Natl. Acad. Sci. USA 2003, 100, 84668471.
Barres, R.; Osler, M.E.; Yan, J.; Rune, A.; Fritz, T.; Caidahl, K.; Krook, A.; Zierath, J.R.
Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial
density. Cell Metab. 2009, 10, 189198.
Barres, R.; Yan, J.; Egan, B.; Treebak, J.T.; Rasmussen, M.; Fritz, T.; Caidahl, K.; Krook, A.;
OGorman, D.J.; Zierath, J.R. Acute exercise remodels promoter methylation in human skeletal
muscle. Cell Metab. 2012, 15, 405411.
Katari, S.; Turan, N.; Bibikova, M.; Erinle, O.; Chalian, R.; Foster, M.; Gaughan, J.P.;
Coutifaris, C.; Sapienza, C. DNA methylation and gene expression differences in children
conceived in vitro or in vivo. Hum. Mol. Genet. 2009, 18, 37693778.
Morgan, H.D.; Sutherland, H.G.; Martin, D.I.; Whitelaw, E. Epigenetic inheritance at the agouti
locus in the mouse. Nat. Genet. 1999, 23, 314318.
Wolff, G.L.; Kodell, R.L.; Moore, S.R.; Cooney, C.A. Maternal epigenetics and methyl
supplements affect agouti gene expression in Avy/a mice. FASEB J. 1998, 12, 949957.
Ng, S.F.; Lin, R.C.; Laybutt, D.R.; Barres, R.; Owens, J.A.; Morris, M.J. Chronic high-fat diet in
fathers programs beta-cell dysfunction in female rat offspring. Nature 2010, 467, 963966.
Godfrey, K.M.; Sheppard, A.; Gluckman, P.D.; Lillycrop, K.A.; Burdge, G.C.; McLean, C.;
Rodford, J.; Slater-Jefferies, J.L.; Garratt, E.; Crozier, S.R.; et al. Epigenetic gene promoter
methylation at birth is associated with childs later adiposity. Diabetes 2011, 60, 15281534.
Bartel, D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004, 116,
281297.
Kallio, P.; Kolehmainen, M.; Laaksonen, D.E.; Kekalainen, J.; Salopuro, T.; Sivenius, K.;
Pulkkinen, L.; Mykkanen, H.M.; Niskanen, L.; Uusitupa, M.; Poutanen, K.S. Dietary carbohydrate
modification induces alterations in gene expression in abdominal subcutaneous adipose tissue in
persons with the metabolic syndrome: The FUNGENUT Study. Am. J. Clin. Nutr. 2007, 85,
14171427.
Zhang, L.; Hou, D.; Chen, X.; Li, D.; Zhu, L.; Zhang, Y.; Li, J.; Bian, Z.; Liang, X.; Cai, X.; et al.
Exogenous plant MIR168a specifically targets mammalian LDLRAP1: Evidence of cross-kingdom
regulation by microRNA. Cell Res. 2012, 22, 107126.
Panagiotou, G.; Nielsen, J. Nutritional systems biology: Definitions and approaches. Ann. Rev.
Nutr. 2009, 29, 329339.
Crujeiras, A.B.; Parra, D.; Milagro, F.I.; Goyenechea, E.; Larrarte, E.; Margareto, J.;
Martinez, J.A. Differential expression of oxidative stress and inflammation related genes in
peripheral blood mononuclear cells in response to a low-calorie diet: A nutrigenomics study.
OMICS 2008, 12, 251261.

Nutrients 2012, 4
83.

84.

85.
86.
87.
88.
89.
90.
91.

92.

93.

94.
95.

96.

97.

98.

1935

Caesar, R.; Manieri, M.; Kelder, T.; Boekschoten, M.; Evelo, C.; Muller, M.; Kooistra, T.;
Cinti, S.; Kleemann, R.; Drevon, C.A. A combined transcriptomics and lipidomics analysis of
subcutaneous, epididymal and mesenteric adipose tissue reveals marked functional differences.
PLoS One 2010, 5, e11525.
Henze, A.; Rohn, S.; Gericke, B.; Raila, J.; Schweigert, F.J. Structural modifications of serum
transthyretin in rats during protein-energy malnutrition. Rapid Commun. Mass Spectrom. 2008,
22, 32703274.
Romagnolo, D.F.; Milner, J.A. Opportunities and challenges for nutritional proteomics in cancer
prevention. J. Nutr. 2012, 142, 1360S1369S.
Kussmann, M.; Panchaud, A.; Affolter, M. Proteomics in nutrition: Status quo and outlook for
biomarkers and bioactives. J. Proteome Res. 2010, 9, 48764887.
Moore, J.B.; Weeks, M.E. Proteomics and systems biology: Current and future applications in
the nutritional sciences. Adv. Nutr. 2011, 2, 355364.
Rabilloud, T. Two-dimensional gel electrophoresis in proteomics: Old, old fashioned, but it still
climbs up the mountains. Proteomics 2002, 2, 310.
Walther, T.C.; Mann, M. Mass spectrometry-based proteomics in cell biology. J. Cell Biol. 2010,
190, 491500.
Domon, B.; Aebersold, R. Options and considerations when selecting a quantitative proteomics
strategy. Nat. Biotechnol. 2010, 28, 710721.
Norheim, F.; Raastad, T.; Thiede, B.; Rustan, A.C.; Drevon, C.A.; Haugen, F. Proteomic
identification of secreted proteins from human skeletal muscle cells and expression in response to
strength training. Am. J. Physiol. Endocrinol. Metab. 2011, 301, E1013E1021.
Unwin, R.D.; Griffiths, J.R.; Whetton, A.D. Simultaneous analysis of relative protein expression
levels across multiple samples using iTRAQ isobaric tags with 2D nano LC-MS/MS. Nat.
Protoc. 2010, 5, 15741582.
Ong, S.E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D.B.; Steen, H.; Pandey, A.; Mann, M.
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach
to expression proteomics. Mol. Cell. Proteomics 2002, 1, 376386.
Gouw, J.W.; Krijgsveld, J.; Heck, A.J. Quantitative proteomics by metabolic labeling of model
organisms. Mol. Cell. Proteomics 2010, 9, 1124.
Forner, F.; Kumar, C.; Luber, C.A.; Fromme, T.; Klingenspor, M.; Mann, M. Proteome
differences between brown and white fat mitochondria reveal specialized metabolic functions.
Cell Metab. 2009, 10, 324335.
Hwang, H.; Bowen, B.P.; Lefort, N.; Flynn, C.R.; de Filippis, E.A.; Roberts, C.; Smoke, C.C.;
Meyer, C.; Hojlund, K.; Yi, Z.; Mandarino, L.J. Proteomics analysis of human skeletal muscle
reveals novel abnormalities in obesity and type 2 diabetes. Diabetes 2010, 59, 3342.
Schwer, B.; Eckersdorff, M.; Li, Y.; Silva, J.C.; Fermin, D.; Kurtev, M.V.; Giallourakis, C.;
Comb, M.J.; Alt, F.W.; Lombard, D.B. Calorie restriction alters mitochondrial protein
acetylation. Aging Cell 2009, 8, 604606.
Picotti, P.; Aebersold, R. Selected reaction monitoring-based proteomics: Workflows, potential,
pitfalls and future directions. Nat. Methods 2012, 9, 555566.

Nutrients 2012, 4
99.
100.

101.
102.

103.
104.
105.

106.
107.

108.

109.

110.

111.
112.
113.

114.

1936

Patti, G.J.; Yanes, O.; Siuzdak, G. Innovation: Metabolomics: The apogee of the omics trilogy.
Nat. Rev. Mol. Cell Biol. 2012, 13, 263269.
Aune, D.; Chan, D.S.; Vieira, A.R.; Navarro Rosenblatt, D.A.; Vieira, R.; Greenwood, D.C.;
Norat, T. Dietary compared with blood concentrations of carotenoids and breast cancer risk:
A systematic review and meta-analysis of prospective studies. Am. J. Clin. Nutr. 2012, 96,
356373.
Gibney, M.J.; Walsh, M.; Brennan, L.; Roche, H.M.; German, B.; van Ommen, B. Metabolomics
in human nutrition: Opportunities and challenges. Am. J. Clin. Nutr. 2005, 82, 497503.
Lodge, J.K. Symposium 2: Modern approaches to nutritional research challenges: Targeted and
non-targeted approaches for metabolite profiling in nutritional research. Proc. Nutr. Soc 2010,
69, 95102.
Zhang, A.; Sun, H.; Wang, P.; Han, Y.; Wang, X. Modern analytical techniques in metabolomics
analysis. Analyst 2012, 137, 293300.
Griffiths, W.J.; Koal, T.; Wang, Y.; Kohl, M.; Enot, D.P.; Deigner, H.P. Targeted metabolomics
for biomarker discovery. Angew. Chem. Int. Ed. Engl. 2010, 49, 54265445.
Karlsen, A.; Svendsen, M.; Seljeflot, I.; Sommernes, M.A.; Sexton, J.; Brevik, A.; Erlund, I.;
Serafini, M.; Bastani, N.; Remberg, S.F.; et al. Compliance, tolerability and safety of
two antioxidant-rich diets: A randomised controlled trial in male smokers. Br. J. Nutr. 2011, 106,
557571.
German, J.B.; Gillies, L.A.; Smilowitz, J.T.; Zivkovic, A.M.; Watkins, S.M. Lipidomics and
lipid profiling in metabolomics. Curr. Opin. Lipidol. 2007, 18, 6671.
Karlsen, A.; Retterstol, L.; Laake, P.; Paur, I.; Bohn, S.K.; Sandvik, L.; Blomhoff, R.
Anthocyanins inhibit nuclear factor-kappaB activation in monocytes and reduce plasma
concentrations of pro-inflammatory mediators in healthy adults. J. Nutr. 2007, 137, 19511954.
Karlsen, A.; Paur, I.; Bohn, S.K.; Sakhi, A.K.; Borge, G.I.; Serafini, M.; Erlund, I.; Laake, P.;
Tonstad, S.; Blomhoff, R. Bilberry juice modulates plasma concentration of NF-kappaB related
inflammatory markers in subjects at increased risk of CVD. Eur. J. Nutr. 2010, 49, 345355.
Brevik, A.; Karlsen, A.; Azqueta, A.; Tirado, A.E.; Blomhoff, R.; Collins, A. Both base excision
repair and nucleotide excision repair in humans are influenced by nutritional factors. Cell
Biochem. Funct. 2011, 29, 3642.
Brevik, A.; Gaivao, I.; Medin, T.; Jorgenesen, A.; Piasek, A.; Elilasson, J.; Karlsen, A.;
Blomhoff, R.; Veggan, T.; Duttaroy, A.K.; Collins, A.R. Supplementation of a western diet with
golden kiwifruits (Actinidia chinensis var.Hort 16A:) effects on biomarkers of oxidation
damage and antioxidant protection. Nutr. J. 2011, 10, 54.
Puri, R.; Duong, M.; Uno, K.; Kataoka, Y.; Nicholls, S.J. The emerging role of plasma
lipidomics in cardiovascular drug discovery. Expert Opin. Drug Discov. 2012, 7, 6372.
Wood, P.L. Lipidomics of Alzheimers disease: Current status. Alzheimers Res. Ther. 2012, 4, 5.
Wittwer, J.; Rubio-Aliaga, I.; Hoeft, B.; Bendik, I.; Weber, P.; Daniel, H. Nutrigenomics in
human intervention studies: Current status, lessons learned and future perspectives. Mol. Nutr.
Food Res. 2011, 55, 341358.
Bocci, V. The neglected organ: Bacterial flora has a crucial immunostimulatory role. Perspect.
Biol. Med. 1992, 35, 251260.

Nutrients 2012, 4

1937

115. Backhed, F.; Ley, R.E.; Sonnenburg, J.L.; Peterson, D.A.; Gordon, J.I. Host-bacterial mutualism
in the human intestine. Science 2005, 307, 19151920.
116. Nicholson, J.K.; Holmes, E.; Kinross, J.; Burcelin, R.; Gibson, G.; Jia, W.; Pettersson, S.
Host-gut microbiota metabolic interactions. Science 2012, 336, 12621267.
117. Hooper, L.V.; Littman, D.R.; Macpherson, A.J. Interactions between the microbiota and the
immune system. Science 2012, 336, 12681273.
118. Suau, A.; Bonnet, R.; Sutren, M.; Godon, J.J.; Gibson, G.R.; Collins, M.D.; Dore, J. Direct
analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular
species within the human gut. Appl. Environ. Microbiol. 1999, 65, 47994807.
119. Zoetendal, E.G.; Rajilic-Stojanovic, M.; de Vos, W.M. High-throughput diversity and functionality
analysis of the gastrointestinal tract microbiota. Gut 2008, 57, 16051615.
120. The Human Microbiome Project Consortium. Structure, function and diversity of the healthy
human microbiome. Nature 2012, 486, 207214.
121. Frank, D.N.; St Amand, A.L.; Feldman, R.A.; Boedeker, E.C.; Harpaz, N.; Pace, N.R.
Molecular-phylogenetic characterization of microbial community imbalances in human
inflammatory bowel diseases. Proc. Natl. Acad. Sci. USA 2007, 104, 1378013785.
122. Tap, J.; Mondot, S.; Levenez, F.; Pelletier, E.; Caron, C.; Furet, J.P.; Ugarte, E.;
Munoz-Tamayo, R.; Paslier, D.L.; Nalin, R.; et al. Towards the human intestinal microbiota
phylogenetic core. Environ. Microbiol. 2009, 11, 25742584.
123. Gill, S.R.; Pop, M.; Deboy, R.T.; Eckburg, P.B.; Turnbaugh, P.J.; Samuel, B.S.; Gordon, J.I.;
Relman, D.A.; Fraser-Liggett, C.M.; Nelson, K.E. Metagenomic analysis of the human distal gut
microbiome. Science 2006, 312, 13551359.
124. Qin, J.; Li, R.; Raes, J.; Arumugam, M.; Burgdorf, K.S.; Manichanh, C.; Nielsen, T.; Pons, N.;
Levenez, F.; Yamada, T.; et al. A human gut microbial gene catalogue established by
metagenomic sequencing. Nature 2010, 464, 5965.
125. Dutton, R.J.; Turnbaugh, P.J. Taking a metagenomic view of human nutrition. Curr. Opin. Clin.
Nutr. Metab. Care 2012, 15, 448454.
126. Wu, G.D.; Chen, J.; Hoffmann, C.; Bittinger, K.; Chen, Y.Y.; Keilbaugh, S.A.; Bewtra, M.;
Knights, D.; Walters, W.A.; Knight, R.; et al. Linking long-term dietary patterns with gut
microbial enterotypes. Science 2011, 334, 105108.
127. Walker, A.W.; Ince, J.; Duncan, S.H.; Webster, L.M.; Holtrop, G.; Ze, X.; Brown, D.;
Stares, M.D.; Scott, P.; Bergerat, A.; et al. Dominant and diet-responsive groups of bacteria
within the human colonic microbiota. ISME J. 2011, 5, 220230.
128. Davis, L.M.; Martinez, I.; Walter, J.; Goin, C.; Hutkins, R.W. Barcoded pyrosequencing reveals
that consumption of galactooligosaccharides results in a highly specific bifidogenic response in
humans. PLoS One 2011, 6, e25200.
129. Ramirez-Farias, C.; Slezak, K.; Fuller, Z.; Duncan, A.; Holtrop, G.; Louis, P. Effect of inulin on
the human gut microbiota: Stimulation of Bifidobacterium adolescentis and Faecalibacterium
prausnitzii. Br. J. Nutr. 2009, 101, 541550.
130. Flint, H.J.; Scott, K.P.; Louis, P.; Duncan, S.H. The role of the gut microbiota in nutrition and
health. Nat. Rev. Gastroenterol. Hepatol. 2012, 9, 577589.
131. Weissleder, R.; Pittet, M.J. Imaging in the era of molecular oncology. Nature 2008, 452, 580589.

Nutrients 2012, 4

1938

132. Heymsfield, S.B. Development of imaging methods to assess adiposity and metabolism. Int. J.
Obes. (Lond.) 2008, 32, S76S82.
133. Rokling-Andersen, M.H.; Rustan, A.C.; Wensaas, A.J.; Kaalhus, O.; Wergedahl, H.; Rost, T.H.;
Jensen, J.; Graff, B.A.; Caesar, R.; Drevon, C.A. Marine n-3 fatty acids promote size reduction of
visceral adipose depots, without altering body weight and composition, in male Wistar rats fed
a high-fat diet. Br. J. Nutr. 2009, 102, 9951006.
134. Wensaas, A.J.; Rustan, A.C.; Rokling-Andersen, M.H.; Caesar, R.; Jensen, J.; Kaalhus, O.;
Graff, B.A.; Gudbrandsen, O.A.; Berge, R.K.; Drevon, C.A. Dietary supplementation of
tetradecylthioacetic acid increases feed intake but reduces body weight gain and adipose depot
sizes in rats fed on high-fat diets. Diabetes Obes. Metab. 2009, 11, 10341049.
135. Seidell, J.C.; Bakker, C.J.; van der Kooy, K. Imaging techniques for measuring adipose-tissue
distributionA comparison between computed tomography and 1.5-T magnetic resonance. Am.
J. Clin. Nutr. 1990, 51, 953957.
136. Klopfenstein, B.J.; Kim, M.S.; Krisky, C.M.; Szumowski, J.; Rooney, W.D.; Purnell, J.Q.
Comparison of 3-T MRI and CT for the measurement of visceral and subcutaneous adipose
tissue in humans. Br. J. Radiol. 2012, 85, e826e830.
137. Gray, C.; MacGillivray, T.J.; Eeley, C.; Stephens, N.A.; Beggs, I.; Fearon, K.C.; Greig, C.A.
Magnetic resonance imaging with k-means clustering objectively measures whole muscle volume
compartments in sarcopenia/cancer cachexia. Clin. Nutr. 2011, 30, 106111.
138. Kielland, A.; Carlsen, H. Molecular imaging of transcriptional regulation during inflammation.
J. Inflamm. (Lond.) 2010, 7, 20.
139. Paur, I.; Balstad, T.R.; Kolberg, M.; Pedersen, M.K.; Austenaa, L.M.; Jacobs, D.R., Jr.;
Blomhoff, R. Extract of oregano, coffee, thyme, clove, and walnuts inhibits NF-kappaB in
monocytes and in transgenic reporter mice. Cancer Prev. Res. (Phila.) 2010, 3, 653663.
140. Zhang, Y.; Proenca, R.; Maffei, M.; Barone, M.; Leopold, L.; Friedman, J.M. Positional cloning
of the mouse obese gene and its human homologue. Nature 1994, 372, 425432.
141. Kaiyala, K.J.; Ramsay, D.S. Direct animal calorimetry, the underused gold standard for
quantifying the fire of life. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2011, 158, 252264.
142. Levine, J.A. Measurement of energy expenditure. Public Health Nutr. 2005, 8, 11231132.
143. Tschop, M.H.; Speakman, J.R.; Arch, J.R.; Auwerx, J.; Bruning, J.C.; Chan, L.; Eckel, R.H.;
Farese, R.V., Jr.; Galgani, J.E.; Hambly, C.; et al. A guide to analysis of mouse energy metabolism.
Nat. Methods 2012, 9, 5763.
144. Butler, A.A.; Kozak, L.P. A recurring problem with the analysis of energy expenditure in genetic
models expressing lean and obese phenotypes. Diabetes 2010, 59, 323329.
145. Azuaje, F.; Devaux, Y.; Wagner, D. Computational biology for cardiovascular biomarker
discovery. Brief. Bioinform. 2009, 10, 367377.
146. Ng, A.; Bursteinas, B.; Gao, Q.; Mollison, E.; Zvelebil, M. Resources for integrative systems
biology: From data through databases to networks and dynamic system models. Brief. Bioinform.
2006, 7, 318330.
147. Evelo, C.T.; van Bochove, K.; Saito, J.T. Answering biological questions: Querying a systems
biology database for nutrigenomics. Genes Nutr. 2011, 6, 8187.

Nutrients 2012, 4

1939

148. Hester, R.L.; Iliescu, R.; Summers, R.; Coleman, T.G. Systems biology and integrative
physiological modelling. J. Physiol. 2011, 589, 10531060.
149. Frayn, K.N. Metabolic Regulation: A Human Perspective. In Metabolic Regulation: A Human
Perspective; Wiley-Blackwell: Oxford, UK, 2010.
150. Rosen, E.D.; Spiegelman, B.M. Adipocytes as regulators of energy balance and glucose
homeostasis. Nature 2006, 444, 847853.
151. Matsuzawa, Y.; Funahashi, T.; Nakamura, T. The concept of metabolic syndrome: Contribution
of visceral fat accumulation and its molecular mechanism. J. Atheroscler. Thromb. 2011, 18,
629639.
152. Fox, C.S.; Liu, Y.; White, C.C.; Feitosa, M.; Smith, A.V.; Heard-Costa, N.; Lohman, K.;
Johnson, A.D.; Foster, M.C.; Greenawalt, D.M.; et al. Genome-wide association for abdominal
subcutaneous and visceral adipose reveals a novel locus for visceral fat in women. PLoS Genet.
2012, 8, e1002695.
153. Vinknes, K.J.; Elshorbagy, A.K.; Drevon, C.A.; Gjesdal, C.G.; Tell, G.S.; Nygrd, O.;
Vollset, S.E.; Refsum, H. Evaluation of the Body Adiposity Index in a Caucasian Population.
The Hordaland Health Study. Am. J. Epidemiol. 2012, in press.
154. Canoy, D. Coronary heart disease and body fat distribution. Curr. Atheroscler. Rep. 2010, 12,
125133.
155. Heitmann, B.L.; Lissner, L. Hip Hip Hurrah! Hip size inversely related to heart disease and total
mortality. Obes. Rev. 2011, 12, 478481.
156. Gesta, S.; Tseng, Y.H.; Kahn, C.R. Developmental origin of fat: Tracking obesity to its source.
Cell 2007, 131, 242256.
157. Cinti, S. Transdifferentiation properties of adipocytes in the adipose organ. Am. J. Physiol.
Endocrinol. Metab. 2009, 297, E977E986.
158. Bjorneboe, A.; Bjorneboe, G.E.; Drevon, C.A. Absorption, transport and distribution of
vitamin E. J. Nutr. 1990, 120, 233242.
159. Pond, C.M. Adipose tissue and the immune system. Prostaglandins Leukot. Essent. Fatty Acids
2005, 73, 1730.
160. Knittle, J.L.; Timmers, K.; Ginsberg-Fellner, F.; Brown, R.E.; Katz, D.P. The growth of adipose
tissue in children and adolescents. Cross-sectional and longitudinal studies of adipose cell
number and size. J. Clin. Invest. 1979, 63, 239246.
161. Seale, P.; Bjork, B.; Yang, W.; Kajimura, S.; Chin, S.; Kuang, S.; Scime, A.; Devarakonda, S.;
Conroe, H.M.; Erdjument-Bromage, H.; et al. PRDM16 controls a brown fat/skeletal muscle
switch. Nature 2008, 454, 961967.
162. Virtanen, K.A.; Lidell, M.E.; Orava, J.; Heglind, M.; Westergren, R.; Niemi, T.; Taittonen, M.;
Laine, J.; Savisto, N.J.; Enerback, S.; Nuutila, P. Functional brown adipose tissue in healthy
adults. N. Engl. J. Med. 2009, 360, 15181525.
163. Cypess, A.M.; Lehman, S.; Williams, G.; Tal, I.; Rodman, D.; Goldfine, A.B.; Kuo, F.C.;
Palmer, E.L.; Tseng, Y.H.; Doria, A.; et al. Identification and importance of brown adipose tissue
in adult humans. N. Engl. J. Med. 2009, 360, 15091517.

Nutrients 2012, 4

1940

164. Van Marken Lichtenbelt, W.D.; Vanhommerig, J.W.; Smulders, N.M.; Drossaerts, J.M.;
Kemerink, G.J.; Bouvy, N.D.; Schrauwen, P.; Teule, G.J. Cold-activated brown adipose tissue in
healthy men. N. Engl. J. Med. 2009, 360, 15001508.
165. Saito, M.; Okamatsu-Ogura, Y.; Matsushita, M.; Watanabe, K.; Yoneshiro, T.; Nio-Kobayashi,
J.; Iwanaga, T.; Miyagawa, M.; Kameya, T.; Nakada, K.; et al. High incidence of metabolically
active brown adipose tissue in healthy adult humans: Effects of cold exposure and adiposity.
Diabetes 2009, 58, 15261531.
166. Kajimura, S.; Seale, P.; Kubota, K.; Lunsford, E.; Frangioni, J.V.; Gygi, S.P.; Spiegelman, B.M.
Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex.
Nature 2009, 460, 11541158.
167. Ahfeldt, T.; Schinzel, R.T.; Lee, Y.K.; Hendrickson, D.; Kaplan, A.; Lum, D.H.; Camahort, R.;
Xia, F.; Shay, J.; Rhee, E.P.; et al. Programming human pluripotent stem cells into white and
brown adipocytes. Nat. Cell Biol. 2012, 14, 209219.
168. Spalding, K.L.; Arner, E.; Westermark, P.O.; Bernard, S.; Buchholz, B.A.; Bergmann, O.;
Blomqvist, L.; Hoffstedt, J.; Naslund, E.; Britton, T.; et al. Dynamics of fat cell turnover in
humans. Nature 2008, 453, 783787.
169. Arner, P.; Bernard, S.; Salehpour, M.; Possnert, G.; Liebl, J.; Steier, P.; Buchholz, B.A.;
Eriksson, M.; Arner, E.; Hauner, H.; et al. Dynamics of human adipose lipid turnover in health
and metabolic disease. Nature 2011, 478, 110113.
170. Simon, G. Histogenesis, Section 5: Adipose Tissue; American Physiological Society:
Washington, DC, USA, 1965.
171. Tang, W.; Zeve, D.; Suh, J.M.; Bosnakovski, D.; Kyba, M.; Hammer, R.E.; Tallquist, M.D.;
Graff, J.M. White fat progenitor cells reside in the adipose vasculature. Science 2008, 322,
583586.
172. Rodeheffer, M.S.; Birsoy, K.; Friedman, J.M. Identification of white adipocyte progenitor cells
in vivo. Cell 2008, 135, 240249.
173. Tran, K.V.; Gealekman, O.; Frontini, A.; Zingaretti, M.C.; Morroni, M.; Giordano, A.;
Smorlesi, A.; Perugini, J.; de Matteis, R.; Sbarbati, A.; et al. The vascular endothelium of the
adipose tissue gives rise to both white and brown fat cells. Cell Metab. 2012, 15, 222229.
174. Gupta, R.K.; Mepani, R.J.; Kleiner, S.; Lo, J.C.; Khandekar, M.J.; Cohen, P.; Frontini, A.;
Bhowmick, D.C.; Ye, L.; Cinti, S.; Spiegelman, B.M. Zfp423 expression identifies committed
preadipocytes and localizes to adipose endothelial and perivascular cells. Cell Metab. 2012, 15,
230239.
175. Drevon, C.A. Fatty acids and expression of adipokines. Biochim. Biophys. Acta 2005, 1740,
287292.
176. Skurk, T.; Alberti-Huber, C.; Herder, C.; Hauner, H. Relationship between adipocyte size and
adipokine expression and secretion. J. Clin. Endocrinol. Metab. 2007, 92, 10231033.
177. Haugen, F.; Zahid, N.; Dalen, K.T.; Hollung, K.; Nebb, H.I.; Drevon, C.A. Resistin expression in
3T3-L1 adipocytes is reduced by arachidonic acid. J. Lipid Res. 2005, 46, 143153.
178. DeFronzo, R.A.; Jacot, E.; Jequier, E.; Maeder, E.; Wahren, J.; Felber, J.P. The effect of insulin
on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral
venous catheterization. Diabetes 1981, 30, 10001007.

Nutrients 2012, 4

1941

179. Jensen, J.; Rustad, P.I.; Kolnes, A.J.; Lai, Y.C. The role of skeletal muscle glycogen breakdown
for regulation of insulin sensitivity by exercise. Front. Physiol. 2011, 2, 112.
180. Jensen, J.; Ruge, T.; Lai, Y.C.; Svensson, M.K.; Eriksson, J.W. Effects of adrenaline on
whole-body glucose metabolism and insulin-mediated regulation of glycogen synthase and PKB
phosphorylation in human skeletal muscle. Metabolism 2011, 60, 215226.
181. Ruderman, N.B.; Xu, X.J.; Nelson, L.; Cacicedo, J.M.; Saha, A.K.; Lan, F.; Ido, Y. AMPK and
SIRT1: A long-standing partnership? Am. J. Physiol. Endocrinol. Metab. 2010, 298, E751E760.
182. Narkar, V.A.; Downes, M.; Yu, R.T.; Embler, E.; Wang, Y.X.; Banayo, E.; Mihaylova, M.M.;
Nelson, M.C.; Zou, Y.; Juguilon, H.; et al. AMPK and PPARdelta agonists are exercise mimetics.
Cell 2008, 134, 405415.
183. Alibegovic, A.C.; Sonne, M.P.; Hojbjerre, L.; Bork-Jensen, J.; Jacobsen, S.; Nilsson, E.;
Faerch, K.; Hiscock, N.; Mortensen, B.; Friedrichsen, M.; et al. Insulin resistance induced by
physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young
men. Am. J. Physiol. Endocrinol. Metab. 2010, 299, E752E763.
184. Giebelstein, J.; Poschmann, G.; Hojlund, K.; Schechinger, W.; Dietrich, J.W.; Levin, K.;
Beck-Nielsen, H.; Podwojski, K.; Stuhler, K.; Meyer, H.E.; Klein, H.H. The proteomic signature
of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased
mitochondrial enzymes. Diabetologia 2012, 55, 11141127.
185. Rowlands, D.S.; Thomson, J.S.; Timmons, B.W.; Raymond, F.; Fuerholz, A.; Mansourian, R.;
Zwahlen, M.C.; Metairon, S.; Glover, E.; Stellingwerff, T.; et al. Transcriptome and translational
signaling following endurance exercise in trained skeletal muscle: Impact of dietary protein.
Physiol. Genomics 2011, 43, 10041020.
186. Van Loon, L.J.; Greenhaff, P.L.; Constantin-Teodosiu, D.; Saris, W.H.; Wagenmakers, A.J. The
effects of increasing exercise intensity on muscle fuel utilisation in humans. J. Physiol. 2001,
536, 295304.
187. Aas, V.; Kase, E.T.; Solberg, R.; Jensen, J.; Rustan, A.C. Chronic hyperglycaemia promotes
lipogenesis and triacylglycerol accumulation in human skeletal muscle cells. Diabetologia 2004,
47, 14521461.
188. Dresner, A.; Laurent, D.; Marcucci, M.; Griffin, M.E.; Dufour, S.; Cline, G.W.; Slezak, L.A.;
Andersen, D.K.; Hundal, R.S.; Rothman, D.L.; et al. Effects of free fatty acids on glucose
transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J. Clin. Invest. 1999, 103,
253259.
189. Helge, J.W.; Kiens, B. Muscle enzyme activity in humans: Role of substrate availability and
training. Am. J. Physiol. 1997, 272, R1620R1624.
190. Pedersen, B.K.; Febbraio, M.A. Muscle as an endocrine organ: Focus on muscle-derived
interleukin-6. Physiol. Rev. 2008, 88, 13791406.
191. McPherron, A.C.; Lawler, A.M.; Lee, S.J. Regulation of skeletal muscle mass in mice by a new
TGF-beta superfamily member. Nature 1997, 387, 8390.
192. Ostrowski, K.; Rohde, T.; Zacho, M.; Asp, S.; Pedersen, B.K. Evidence that interleukin-6 is
produced in human skeletal muscle during prolonged running. J. Physiol. 1998, 508, 949953.

Nutrients 2012, 4

1942

193. Haugen, F.; Norheim, F.; Lian, H.; Wensaas, A.J.; Dueland, S.; Berg, O.; Funderud, A.;
Skalhegg, B.S.; Raastad, T.; Drevon, C.A. IL-7 is expressed and secreted by human skeletal
muscle cells. Am. J. Physiol. Cell Physiol. 2010, 298, C807C816.
194. Nielsen, A.R.; Hojman, P.; Erikstrup, C.; Fischer, C.P.; Plomgaard, P.; Mounier, R.;
Mortensen, O.H.; Broholm, C.; Taudorf, S.; Krogh-Madsen, R.; et al. Association between
interleukin-15 and obesity: Interleukin-15 as a potential regulator of fat mass. J. Clin.
Endocrinol. Metab. 2008, 93, 44864493.
195. Nitert, M.D.; Dayeh, T.; Volkov, P.; Elgzyri, T.; Hall, E.; Nilsson, E.; Yang, B.T.; Lang, S.;
Parikh, H.; Wessman, Y.; et al. Impact of an exercise intervention on DNA methylation in
skeletal muscle from first-degree relatives of patients with type 2 diabetes. Diabetes 2012,
doi:10.2337/db11-1653.
196. Bostrom, P.; Wu, J.; Jedrychowski, M.P.; Korde, A.; Ye, L.; Lo, J.C.; Rasbach, K.A.;
Bostrom, E.A.; Choi, J.H.; Long, J.Z.; et al. A PGC1-alpha-dependent myokine that drives
brown-fat-like development of white fat and thermogenesis. Nature 2012, 481, 463468.
197. Vallim, T.; Salter, A.M. Regulation of hepatic gene expression by saturated fatty acids.
Prostaglandins Leukot. Essent. Fatty Acids 2010, 82, 211218.
198. Krawczyk, M.; Bonfrate, L.; Portincasa, P. Nonalcoholic fatty liver disease. Best Pract. Res.
Clin. Gastroenterol. 2010, 24, 695708.
199. Takamura, T.; Misu, H.; Ota, T.; Kaneko, S. Fatty liver as a consequence and cause of insulin
resistance: Lessons from type 2 diabetic liver. Endocr. J. 2012, 59, 745763.
200. Thomas, E.L.; Parkinson, J.R.; Frost, G.S.; Goldstone, A.P.; Dore, C.J.; McCarthy, J.P.;
Collins, A.L.; Fitzpatrick, J.A.; Durighel, G.; Taylor-Robinson, S.D.; Bell, J.D. The missing risk:
MRI and MRS phenotyping of abdominal adiposity and ectopic fat. Obesity (Silver Spring) 2012,
20, 7687.
201. Chalasani, N.; Younossi, Z.; Lavine, J.E.; Diehl, A.M.; Brunt, E.M.; Cusi, K.; Charlton, M.;
Sanyal, A.J. The diagnosis and management of non-alcoholic fatty liver disease: Practice
Guideline by the American Association for the Study of Liver Diseases, American College of
Gastroenterology, and the American Gastroenterological Association. Hepatology 2012, 55,
20052023.
202. Valenzuela, R.; Videla, L.A. The importance of the long-chain polyunsaturated fatty acid n-6/n-3
ratio in development of non-alcoholic fatty liver associated with obesity. Food Funct. 2011, 2,
644648.
203. Bjermo, H.; Iggman, D.; Kullberg, J.; Dahlman, I.; Johansson, L.; Persson, L.; Berglund, J.;
Pulkki, K.; Basu, S.; Uusitupa, M.; et al. Effects of n-6 PUFAs compared with SFAs on liver fat,
lipoproteins, and inflammation in abdominal obesity: A randomized controlled trial. Am. J. Clin.
Nutr. 2012, 95, 10031012.
204. Farese, R.V., Jr.; Zechner, R.; Newgard, C.B.; Walther, T.C. The problem of establishing
relationships between hepatic steatosis and hepatic insulin resistance. Cell Metab. 2012, 15,
570573.
205. Misu, H.; Takamura, T.; Takayama, H.; Hayashi, H.; Matsuzawa-Nagata, N.; Kurita, S.;
Ishikura, K.; Ando, H.; Takeshita, Y.; Ota, T.; et al. A liver-derived secretory protein,
selenoprotein P, causes insulin resistance. Cell Metab. 2010, 12, 483495.

Nutrients 2012, 4

1943

206. Tierney, A.C.; McMonagle, J.; Shaw, D.I.; Gulseth, H.L.; Helal, O.; Saris, W.H.; Paniagua, J.A.;
Golabek-Leszczynska, I.; Defoort, C.; Williams, C.M.; et al. Effects of dietary fat modification
on insulin sensitivity and on other risk factors of the metabolic syndromeLIPGENE:
A European randomized dietary intervention study. Int. J. Obes. (Lond.) 2011, 35, 800809.
207. Brevik, A.; Andersen, L.F.; Karlsen, A.; Trygg, K.U.; Blomhoff, R.; Drevon, C.A.
Six carotenoids in plasma used to assess recommended intake of fruits and vegetables in
a controlled feeding study. Eur. J. Clin. Nutr. 2004, 58, 11661173.
208. Andersen, L.F.; Solvoll, K.; Drevon, C.A. Very-long-chain n-3 fatty acids as biomarkers for
intake of fish and n-3 fatty acid concentrates. Am. J. Clin. Nutr. 1996, 64, 305311.
209. Singh, B.; Saxena, A. Surrogate markers of insulin resistance: A review. World J. Diabetes 2010,
1, 3647.
210. World Health Organization; International Diabetes Federation. Definition and Diagnosis of
Diabetes Mellitus and Intermediate Hyperglycemia: Report of a WHO/IDF Consultation; World
Health Organization: Geneva, Switerzland, 2006; p. 21.
211. Monzillo, L.U.; Hamdy, O. Evaluation of insulin sensitivity in clinical practice and in research
settings. Nutr. Rev. 2003, 61, 397412.
212. Matsuda, M. Measuring and estimating insulin resistance in clinical and research settings. Nutr.
Metab. Cardiovasc. Dis. 2010, 20, 7986.
213. Lorenzo, C.; Hazuda, H.P.; Haffner, S.M. Insulin resistance and excess risk of diabetes in
Mexican-Americans: The San Antonio Heart Study. J. Clin. Endocrinol. Metab. 2012, 97,
793799.
214. Shaham, O.; Wei, R.; Wang, T.J.; Ricciardi, C.; Lewis, G.D.; Vasan, R.S.; Carr, S.A.;
Thadhani, R.; Gerszten, R.E.; Mootha, V.K. Metabolic profiling of the human response to
a glucose challenge reveals distinct axes of insulin sensitivity. Mol. Syst. Biol. 2008, 4, 214.
215. Wopereis, S.; Rubingh, C.M.; van Erk, M.J.; Verheij, E.R.; van Vliet, T.; Cnubben, N.H.;
Smilde, A.K.; van der Greef, J.; van Ommen, B.; Hendriks, H.F. Metabolic profiling of the
response to an oral glucose tolerance test detects subtle metabolic changes. PLoS One 2009,
4, e4525.
216. Rubio-Aliaga, I.; Roos, B.; Duthie, S.; Crosley, L.; Mayer, C.; Horgan, G.; Colquhoun, I.;
Gall, G.; Huber, F.; Kremer, W.; et al. Metabolomics of prolonged fasting in humans reveals new
catabolic markers. Metabolomics 2010, 7, 375387.
217. Krug, S.; Kastenmuller, G.; Stuckler, F.; Rist, M.J.; Skurk, T.; Sailer, M.; Raffler, J.;
Romisch-Margl, W.; Adamski, J.; Prehn, C.; et al. The dynamic range of the human metabolome
revealed by challenges. FASEB J. 2012, 26, 26072619.
218. Speakman, J.R.; Levitsky, D.A.; Allison, D.B.; Bray, M.S.; de Castro, J.M.; Clegg, D.J.;
Clapham, J.C.; Dulloo, A.G.; Gruer, L.; Haw, S.; et al. Set points, settling points and some
alternative models: Theoretical options to understand how genes and environments combine to
regulate body adiposity. Dis. Model. Mech. 2011, 4, 733745.
219. Hession, M.; Rolland, C.; Kulkarni, U.; Wise, A.; Broom, J. Systematic review of randomized
controlled trials of low-carbohydrate vs. low-fat/low-calorie diets in the management of obesity
and its comorbidities. Obes. Rev. 2009, 10, 3650.

Nutrients 2012, 4

1944

220. Sacks, F.M.; Bray, G.A.; Carey, V.J.; Smith, S.R.; Ryan, D.H.; Anton, S.D.; McManus, K.;
Champagne, C.M.; Bishop, L.M.; Laranjo, N.; et al. Comparison of weight-loss diets with
different compositions of fat, protein, and carbohydrates. N. Engl. J. Med. 2009, 360, 859873.
221. Lagiou, P.; Sandin, S.; Lof, M.; Trichopoulos, D.; Adami, H.O.; Weiderpass, E. Low
carbohydrate-high protein diet and incidence of cardiovascular diseases in Swedish women:
Prospective cohort study. BMJ 2012, 344, e4026.
222. Ericson, U.; Sonestedt, E.; Gullberg, B.; Hellstrand, S.; Hindy, G.; Wirfalt, E.; Orho-Melander, M.
High intakes of protein and processed meat associate with increased incidence of type 2 diabetes.
Br. J. Nutr. 2012, in press.
223. Knowler, W.C.; Fowler, S.E.; Hamman, R.F.; Christophi, C.A.; Hoffman, H.J.; Brenneman,
A.T.; Brown-Friday, J.O.; Goldberg, R.; Venditti, E.; Nathan, D.M. 10-Year follow-up of
diabetes incidence and weight loss in the Diabetes Prevention Program Outcomes Study. Lancet
2009, 374, 16771686.
224. Li, G.; Zhang, P.; Wang, J.; Gregg, E.W.; Yang, W.; Gong, Q.; Li, H.; Li, H.; Jiang, Y.;
An, Y.; et al. The long-term effect of lifestyle interventions to prevent diabetes in the China
Da Qing Diabetes Prevention Study: A 20-year follow-up study. Lancet 2008, 371, 17831789.
225. Lindstrom, J.; Ilanne-Parikka, P.; Peltonen, M.; Aunola, S.; Eriksson, J.G.; Hemio, K.;
Hamalainen, H.; Harkonen, P.; Keinanen-Kiukaanniemi, S.; Laakso, M.; et al. Sustained
reduction in the incidence of type 2 diabetes by lifestyle intervention: Follow-up of the Finnish
Diabetes Prevention Study. Lancet 2006, 368, 16731679.
226. Helland, I.B.; Smith, L.; Saarem, K.; Saugstad, O.D.; Drevon, C.A. Maternal supplementation
with very-long-chain n-3 fatty acids during pregnancy and lactation augments childrens IQ at
4 years of age. Pediatrics 2003, 111, e39e44.
2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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